Anti-ilt7 antibody

ABSTRACT

An antibody binding to IPC was obtained by using an animal cell in which a cell membrane protein associatable with ILT7 was co-expressed as an immunogen. The antibody of the invention has a high specificity which allows immunological distinction between other ILT family molecules and ILT7. The anti-ILT7 antibody of the invention bound to IPC and inhibited the activity thereof. With the anti-ILT7 antibody of the invention, the IPC activity can be inhibited and an interferon-related disease can be treated or prevented. ILT7 expression is maintained even in IPC in the presence of IFNα. Therefore, an inhibitory action of IPC activity by the anti-ILT7 antibody can be expected even in an autoimmune disease patient with an increased production of IFNα.

TECHNICAL FIELD

The present invention relates to an antibody which binds to human ILT7.

BACKGROUND ART

Interferon α (IFNα: hereinafter, “interferon” is abbreviated as IFN) andinterferon β (IFNβ) are known as type 1 IFNs which possess antiviralactivity or antitumor activity. On the other hand, it has also beenrevealed that IFNα is related to autoimmune disease. For example,abnormal production of IFNα has been reported in patients with thefollowing autoimmune diseases. It has also been suggested that symptomsof the autoimmune diseases can be reduced by neutralization of IFNα.

Systemic lupus erythematosus (Shiozawa et al., Arthr. & Rheum. 35, 412,1992)

Chronic rheumatism (Hopkins et al., Clin. Exp. Immunol. 73, 88, 1988)

Cases in which symptoms of the autoimmune diseases had been manifestedor worsened by administration of recombinant IFNα2 or IFN were reported(Wada et al., Am. J. Gastroenterol. 90, 136, 1995; Perez et al., Am. J.Hematol. 49, 365, 1995; Wilson L E et al, Semin Arthritis. Rheum. 32,163-173, 2002.).

Further, it has also been revealed that IFNα induces differentiation ofdendritic cells. The dendritic cell is also an antigen presenting cell.Therefore, it is considered that the differentiation induction ofdendritic cells consists an important mechanism in autoimmune diseases.It has been suggested that there is a deep association between thedifferentiation induction of dendritic cells of IFNα and the onset ofsystemic lupus erythematosus (Blanco et al., Science, 16:294, 1540-1543,2001). Thus, it has been pointed out that IFNα is closely related to theantitumor activity as well as autoimmune diseases. In addition, IFNα isdeeply involved in the onset of psoriasis (Nestle F O et al., J. Exp.Med. 202, 135-143, 2005).

Interferon Producing cells (IPCs) were identified as cells which producetype 1 IFN in large quantities associated with virus infection. Few IPCsare presented in the blood. It is considered that peripheral bloodlymphocytes account for 1% or less of IPCs. However, IPCs have a veryhigh capacity to produce IFN. IFN producing capacity of IPCs reaches,for example, 3000 pg/mL/10⁴ cells. That is, it may be said that most ofthe IFNα or IFNβ in the blood, which is produced at viral infection, isresulted from IPCs, although there are few cells.

On the other hand, IPCs are undifferentiated lymphoid dendritic cellswhich are considered as precursor cells of dendritic cells. IPCs may bereferred to as Plasmacytoid dendritic cells. IPCs are differentiatedinto dendritic cells by virus stimulation and induce the production ofIFNγ or IL-10 by T cells. IPCs are also differentiated into dendriticcells by IL-3 stimulation. The differentiated dendritic cells by IL-3stimulation induce the production of Th2 cytokine (IL-4, IL-5, andIL-10) by T cells. Thus, IPCs have properties which allow them to bedifferentiated into distinct dendritic cells by different stimulation.

Accordingly, IPCs have two profiles: IFN producing cells and precursorcells of dendritic cells. Both cells play an important role in immunesystem. In other words, IPC is one of the important cells which supportimmune system in various aspects.

-   Non-patent document 1: Shiozawa et al., Arthr. & Rheum. 35, 412,    1992-   Non-patent document 2: Hopkins et al., Clin. Exp. Immunol. 73, 88,    1988-   Non-patent document 3: Wada et al., Am. J. Gastroenterol. 90, 136,    1995-   Non-patent document 4: Parez et al., Am. J. Hematol. 49, 365, 1995-   Non-patent document 5: Bianco et al., Science, 16:294, 1540-1543,    2001-   Non-patent document 6: Ju et al., Gene. 2004 Apr. 28; 331: 159-64.-   Non-patent document 7: Colonna M et al., Seminars in Immunology 12:    121-127, 2000.-   Non-patent document 8: Nakajima H. et al., J. Immunology 162: 5-8.    1999-   Non-patent document 9: Wilson L E et al, Semin Arthritis. Rheum. 32,    163-173, 2002-   Non-patent document 10: Nestle F O et al., J. Exp. Med. 202,    135-143, 2005-   Patent-document 1: WO03/12061 (U.S. Patent Published Application No.    2003-148316)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

An objective of the present invention is to provide an antibody bindingto Immunoglobulin-Like transcript-7 (ILT7), and to detect, identify, orisolate IPCs. Another objective of the present invention is to regulatethe activity of IPCs.

Means for Solving the Problems

In order to regulate activity of a humoral factor such as IFN,administration of antibodies, which recognize the factor, is effective.For example, the attempt to treat autoimmune diseases by antibodiesagainst interleukin (IL)-1 or IL-4 have been realized (Guler et al.,Arthritis Rheum., 44. S307, 2001). Further, it is assumed thatneutralizing antibodies can serve as therapeutic agents of autoimmunediseases as with interferon (Stewart, T A. Cytokine Growth Factor Rev.14; 139-154, 2003). It can be predicted that the same approach asdescribed above is effective on IFN produced by IPCs. However, such anapproach is based on the inhibition of effect of humoral factor afterproduction of the factor. If the production of the desired humoralfactor can be directly controlled, more substantial therapeutic effectscan be achieved.

Antibodies, which recognize human IPC, have been reported. For example,anti-BDCA-2 monoclonal antibody is human IPC-specific monoclonalantibody (Dzionek A. et al. J. Immunol. 165: 6037-6046, 2000). It isfound that anti-BDCA-2 monoclonal antibody is effective in inhibitingIFN production by human IPCs (J. Exp. Med. 194: 1823-1834, 2001.). Inaddition, it has also been reported that monoclonal antibodies, whichrecognize interferon-producing cells in mice, inhibit the production ofinterferon (Blood 2004 Jun. 1; 103/11: 4201-4206. Epub 2003 December).It was reported that the reduced number of dendritic cells was due tomonoclonal antibodies against plasmacytoid dendritic cells in mice (J.Immunol. 2003, 171: 6466-6477).

Similarly, if antibodies which recognize human IPCs and can regulate theactivity are provided, it will be useful. For example, the presentinventors have already shown that an antibody, which recognizes Ly49Q,specifically binds to mouse IPCs. However, the antibody against Ly49Qdid not interfere with the activity of mouse IPCs (Blood, 1 Apr. 2005,Vol. 105, No. 7, and pp. 2787-2792; WO2004/13325). On the other hand,ILT7 is known as a molecule whose specific expression is seen inPlasmacytoid dendritic cells (Ju X S et al. and Gene. 2004 Apr. 28; 331:159-64; WO03/12061). However, any antibodies against ILT7 have not beenobtained. Therefore, the effects of antibodies on IPCs are also unknown.

ILT7 is a membrane protein containing an immunoglobulin-like motif. Ithas been reported as one of the molecules expressed in cells of themyeloid system or lymphatic system (Colonna M et al., Seminars inImmunology 12:121-127, 2000.). A plurality of molecules with structuresanalogous to ILT7 is referred to as ILT family. ILT family is alsostructurally similar to killer cell inhibitory receptors (KIR). ILT7 hasfour C-type immunoglobulin-like domains as with other molecules of ILTfamily. It is considered that ILT7 sends activation signals into thecell as with ILT1, ILT1-like protein, ILT8, and LIR6a. It has beenconfirmed that a molecule belonging to ILT family is expressed inhemocyte system cells (Young et al., Immunogenetics 53: 270-278, 2001;“The KIR Gene Cluster. “Carrington, Mary and Norman, Paul. Bethesda(Md.): National Library of Medicine (US), NCBI; 2003).

Then, a high expression of ILT7 was detected in Plasmacytoid dendriticcells (PDC) and a low expression of ILT7 was detected inmonocyte-derived dendritic cells (MDDC) by subtractive hybridization.ILT2 and ILT3 were expressed in not only PDC but also DC obtained fromMDDC or CD34 positive cells. However, since mRNA in ILT7 wasspecifically expressed in PDC, it was found that the mRNA might serve asa marker of PDC. Additionally, it was found that at that time, theexpression of ILT7 was reduced by stimulation of CpG (Ju X S et al.Gene. 2004 Apr. 28; 331: 159-64; WO03/12061).

The present inventors confirmed that specific expression of ILT7 in IPCwas facilitated through the study on human IPC. Then, the presentinventors attempted to produce antibodies of ILT7 and to elucidate theeffects. For example, molecules constituting ILT families such as ILT2and ILT3 have high conservation, particularly in amino acid sequences ofextracellular domains (FIG. 9). These ILT families exhibitcharacteristic expression profiles in various blood cells, respectively.Therefore, it is a very important subject to obtain an antibody whichcan immunologically distinguish between other ILT family molecules andILT7. However, in fact, it was difficult to produce an antibody whichbinds specifically to human IPCs using ILT7 as an immunogen because ofthe obstacles described below.

Generally, a protein produced by gene-recombination technology is usedas an immunogen in order to obtain an antibody which recognizes a traceamount of proteins derived from living organisms. The present inventorstried to express human ILT7 on the basis of information of a basesequence of cDNA of human ILT7, which had already been found, and theamino acid sequence coded by the base sequence (GenBank Accession No.NM_012276). However, the present inventors could not produce human ILT7as a recombinant under normal conditions.

The partial amino acid sequence of natural protein is often tried to beused as an immunogen in order to obtain a protein antibody. However,there are few amino acid sequences specific to human ILT7 in proteinssince homology to the amino acid sequences is extremely high in ILTfamily. In addition, it is necessary to select the region constituted ofthe portion that is recognized as an epitope by antibodies on thesurface of cells for the purpose of allowing antibodies to recognizemolecules on the surface of cells. Therefore, it has been consideredthat formation of an antibody which is specific to ILT7 by using afragment amino acid sequence as an immunogen is not realistic.

The present inventors showed that an antibody, which binds to IPCs,could be obtained by using a special immunogen under such conditions.Further, the present inventors found that the antibody thus obtainedspecifically recognized human IPCs and further had an effect ofregulating the activity and thereby succeeded in completing the presentinvention. That is, the present invention relates to the followinganti-ILT7 antibody, production method thereof, and use thereof.

Effects of the Invention

The present invention provides an immunogen useful in producing anantibody which recognizes human ILT7 and a production method ofanti-human ILT7 antibody using the immunogen. ILT7 is a membrane proteinbelonging to ILT family. Particularly, the amino acid sequence of theextracellular region is highly conserved among ILT families. Therefore,it is extremely difficult to produce an antibody which distinguishesbetween ILT families by general immunization methods. The presentinventors showed that the antibody, which recognizes human ILT7, can beeasily obtained by using animal cells in which ILT7 is coexpressed withcell membrane protein. Anti-ILT7 antibody, which can be obtained by thepresent invention, has a high specificity which distinguishes cellsexpressing other ILT families from those expressing human IPCs.

In a preferred embodiment, anti-human ILT7 antibody provided by thepresent invention binds to human IPCs. In addition, the antibody of thepresent invention specifically recognizes human IPCs. Therefore, it isuseful in detecting and isolating IPCs. IPC is a cell which producesmost of the type 1 interferon. Therefore, the detection and isolationare important in diagnosis and study of diseases that involve IPCs suchas autoimmune diseases. Particularly, according to the findings of thepresent inventors, the expression of ILT7 in IPCs is not reduced underthe presence of IFNα. IFNα expression is often facilitated in patientswith autoimmune diseases. This means that anti-ILT7 antibody of thepresent invention can be used for the detection and isolation of IPCs asto the patients with autoimmune diseases in which the expression of IFNαis facilitated.

Anti-ILT7 antibody provided by the present invention has an effect whichregulates the activity of human IPCs in a preferred embodiment.Therefore, the anti-ILT7 antibody of the present invention can be usedto inhibit the activity of IPCs. As described previously, the expressionof ILT7 in IPCs is not reduced under the presence of IFNα. Therefore, ifthe inhibition of the activity of IPCs by the antibody of the presentinvention is used, a therapeutic effect on the patients with autoimmunediseases in which the expression of IFNα is facilitated may be expected.

Scant IPCs produce a large amount of IFN. Antibodies as many as IFNmolecules are necessary for neutralization of IFN. However, producingcell activation is directly inhibited in the present invention. As aresult, a strong inhibitory effect on IFN can be expected even ifsmaller amount of antibodies are used compared with neutralization byanti-IFN antibody. Furthermore, in the case where IFN is continuouslyproduced, it is predicted that neutralization by IFN antibodies istransient inhibition. In the present invention, since the activity ofIPCs is inhibited, IFN production inhibiting effect can be expected overa long period of time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a photograph in which the expression of mRNA of ILT7 gene isexamined by RT-PCR method. It is a result of the analyzed expression ofmRNA of ILT7 gene in human immunocytes.

FIG. 1b is a diagram in which the expression of mRNA of ILT7 gene invarious human tissues and cells is compared and examined usingquantitative PCR method. The horizontal axis shows the examined tissuesand cells and the vertical axis shows the expression level of ILT7,which is standardized according to the expression level of GAPDH gene.

FIG. 2 is a diagram showing structures of ILT7 protein, where FIG. 2(a)shows an amino acid sequence of ILT7 protein and further shows theestimated secretion signal sequence and transmembrane domain in thedrawing, and FIG. 2(b) shows a schematic diagram of ILT7 proteins thatare encoded by constructed expression vectors.

FIG. 3 is a diagram showing a result that ILT7 expression vector andFcRγ expression vector were introduced into cells and the cell-surfaceexpression of ILT7 molecules was examined by FCM. The horizontal axisshows the fluorescence intensity detected in anti-FLAG antibody, namely,the intensity of cell-surface expression of ILT7 molecules to which FLAGtag was attached and the vertical axis shows the number of cells.

FIG. 4 shows photographs in which ILT7 expression vector and FcRγexpression vector were introduced into cells and the association ofmolecules was analyzed by immunoprecipitation and Western blotting. Theleft side diagrams show results that ILT7 molecule was blotted withanti-FLAG antibody after immunoprecipitating FcRγ molecule with anti-mycantibody (the drawing above) and FcRγ molecule was blotted with anti-mycantibody (the drawing below). Similarly, the right side diagrams showresults that ILT7 molecule was blotted with anti-FLAG antibody afterimmunoprecipitating FcRγ molecule with anti-FLAG antibody (above) andFcRγ molecule was blotted with anti-myc antibody (below).

FIG. 5 is a photograph in which glycosylation of ILT7 molecule wasexamined by introduction of ILT7 expression vector and FcRγ expressionvector into the cell and N-glycosidase treatment. The left side of thephotograph shows the size of ILT7 in the case where ILT7 was not treatedwith N-glycosidase and the right side of the photograph shows the sizeof ILT7 in the case where N-glycosidase treatment was performed.

FIG. 6a is a diagram in which the responsiveness of the producedanti-ILT7 monoclonal antibody was examined by FCM analysis. (a) shows aresult that binding of anti-ILT7 antibody to IPC fraction of BDCA-2positive was analyzed by using human peripheral blood lymphocytes anddouble staining with the anti-ILT7 antibody and anti-BDCA-2 antibody.The vertical axis shows the responsiveness to BDCA-2 antibody and thehorizontal axis shows the responsiveness to each of the producedanti-ILT7 antibodies.

FIG. 6b is a diagram in which the responsiveness of the producedanti-ILT7 monoclonal antibodies was examined by FCM analysis. (b) showsa result in which binding of anti-ILT7 antibody to ILT7 molecule wasexamined by using 293T cells into which ILT7 and FcRγ expression vectorshad been introduced. The vertical axis shows the responsiveness ofanti-FLAG antibody, namely, the intensity of expression of ILT7molecules to which FLAG tag was attached and the horizontal axis showsthe responsiveness of respective anti-ILT7 antibodies.

FIG. 7 is a diagram in which among the produced anti-ILT7 monoclonalantibodies, the responsiveness of two clones to human peripheral bloodlymphocytes was examined by FCM analysis. Three graphs on the left showsthe results of #11 and three graphs on the right shows the results of#17. In the left side diagrams, each axis with the mark of ILT7 showsthe responsiveness of ILT7#11. Similarly, in the right side diagrams,each axis with the mark of ILT7 shows the responsiveness of ILT7#17.

FIG. 8 is a result in which binding activity of the produced anti-ILT7monoclonal antibodies ILT7#11 and ILT7#17 to human lymphocytes wascompared with that of anti-BDCA-2 antibody and examined. The verticalaxis shows the responsiveness of anti-CD123 antibody and the horizontalaxis shows the responsiveness of each antibody. That is, each antibodybinds to a portion of CD123 positive cell. It is a diagram showing theresults in which the responsiveness was analyzed when lymphocyte cellswere stimulated by two kinds of CpGs and IFNα.

FIG. 9a is a diagram showing amino acid sequences of family moleculeswith high homology to ILT7 molecules. Each amino acid sequence of theextracellular region is mainly shown as an alignment; FIG. 9b is acontinuation of FIG. 9a ; and FIG. 9c is a continuation of FIG. 9 b.

FIG. 10 is a result in which the responsiveness of the producedanti-ILT7 monoclonal antibodies ILT7#11 and ILT7#17 to ILT1, ILT2, andILT3 molecules was examined using cells into which their expressionvectors were introduced. The upper diagram shows the results where theresponsiveness to cells in which ILT7 molecules with a FLAG tag had beencoexpressed with FcRγ was reaffirmed. The lower diagram shows theresults where the responsiveness to cells into which ILT1, ILT2, ILT3,and FcRγ were introduced (left diagram: ILT7#11, right diagram:ILT7#17). The horizontal axis shows the responsiveness of each anti-ILT7antibody.

FIG. 11 is a diagram showing the effect of the produced anti-ILT7monoclonal antibodies ILT7#11 and ILT7#17 on the interferogenic capacityof human lymphocytes. In the diagram, the horizontal axis shows IFNαconcentration in a culture supernatant when human lymphocytes werestimulated by influenza virus and the vertical axis shows the treatedantibodies. The term “no infections” indicates the results of cellswhich were not stimulated by influenza virus.

FIG. 12 is a diagram showing CDC activity of the produced anti-ILT7monoclonal antibodies ILT7#37, ILT7#28, and ILT7#33. Even when anti-ILT7monoclonal antibodies obtained from any hybridoma were used, 80% or moreof CDC activity was exhibited at the antibody concentration of 0.1 μg/mlor higher. In the case of antibodies other than anti-ILT7 monoclonalantibody, CDC activity to target cells was not observed.

FIG. 13 is a diagram showing internalization to target cells of theproduced anti-ILT7 monoclonal antibodies ILT7#17, ILT7#26, ILT7#37,ILT7#28, and ILT7#33.

The fluorescence intensity of APC is an indicator of the amount ofILT7-anti-ILT7 antibody immune complex which was present on the surfaceof cells before incubation and it is detected regardless of whetherILT7-anti-ILT7 antibody immune complex is present on the target cellsurface or is incorporated into the cell after incubation. On the otherhand, the fluorescence intensity of FITC is an indicator of the amountof ILT7-anti-ILT7 antibody immune complex which remains on the surfaceof cells after incubation. That is, the fluorescence intensity of FITCis decreased by internalization.

BEST MODE FOR CARRYING OUT THE INVENTION

It has been reported that human ILT7 (immunoglobulin-like transcript-7)is a molecule which is specifically expressed in Plasmacytoid dendriticcells (Gene. 2004 Apr. 28; 331:1 59-64; WO03/12061). Alternatively, itis also known that human ILT7 can be used as a predictive indicator forprognosis of lymphoma (WO2005/24043). However, a method for producing anantibody capable of recognizing human ILT7 has not been established.

Human ILT7 consists of 499 amino acid residues as shown in SEQ ID NO: 2and it is a type 1 transmembrane protein comprising fourimmunoglobulin-like domains in the structure and one transmembraneregion (445-466; from 429 to 450 in SEQ ID NO: 2). Among 444 amino acidresidues including N-terminal, 16 amino acid residues (from −15 to −1,in SEQ ID NO: 2) are signal sequences and 17 to 444 amino acid residues(from 1 to 428, in SEQ ID NO: 2) constitute an extracellular domain. Onthe other hand, the C-terminal region is an intracellular domain. Mostof portions of the human ILT7 are extracellular domains and 33 aminoacid residues constitute an intracellular domain (from 467 to 499; from451 to 483, in SEQ ID NO: 2). It is not predicted that a motif, which isinvolved in signalization, is present in an intracellular domain. A fulllength amino acid sequence of human ILT7 is shown in SEQ ID NO: 2 and abase sequence of cDNA encoding the amino acid sequence is shown in SEQID NO: 1. Here, the coding regions of the matured peptide (72) . . .(1520), shown in SEQ ID NO: 1, do not comprise the termination andinitiation codons. That is, protein coding sequences which comprise thetermination and initiation codons in SEQ ID NO: 1 are from 24 to 1523.

It is considered that the ligand signal is transmitted to cells byassociation of human ILT7 with a signal-transducing molecule. Forexample, most of the Fc receptor γ-chains are present in cells. Inaddition, the intracellular domain contains an immunoreceptortyrosine-based activation motif (ITAM) which is involved insignalization. ITAM is an amino acid sequence portion, which is commonlyseen in adaptor molecules that are associated with immunoreceptors suchas Fc receptors. A motif such as YxxL (SEQ ID NO: 76), which is a targetof tyrosine phosphorylation, is comprised in ITAM and the signal istransmitted by the phosphorylation. Known examples of thesignal-transducing molecule, which comprises ITAM in an intracellulardomain, include CD3ζ and DAP12 in addition to Fc receptor γ-chain. Amongthese signal-transducing molecules, the molecule associated with humanILT7 is predicted to be the Fc receptor γ-chain. Currently, a ligand,which binds to human ILT7, has not been found.

The present inventors confirmed that ILT7 was specifically expressed inhuman IPCs by gene expression analysis. The present inventors consideredthat it would be useful in the study of IPCs if an antibody capable ofdistinguishing human ILT7 from other molecules immunologically could beobtained. However, many molecules with similar structures exist in ILTfamily including ILT7. Molecules such as ILT1, ILT2, ILT3, ILT4, ILT5,ILT6, or LIR-8 comprise highly homologous amino acid sequences,particularly in their extracellular domains. Therefore, the presentinventors considered that it was difficult to obtain an antibody capableof distinguishing between these molecules using a domain peptidecomprising a partial amino acid sequence which constitute anextracellular domain as an immunogen. Then, the present inventors havetried to produce an antibody against human ILT7 using the cellsexpressing human ILT7 as immunogens.

However, the use of general expression vectors did not cause theexpression of cDNA of human ILT7 in animal cells. It has been reportedthat ILT1 molecule having a structure very similar to ILT7 associateswith the Fc receptor γ-chain. That is, when cells in which the Fcreceptor γ-chain was expressed such as RBL (rat basophilic leukemia)cells and P815 (mouse mastocytoma) cells were used as host cells, theexpression of ILT1 on the cell surface was observed. However, if ILT1was forced to be expressed in 293 cells in which Fc receptor γ-chain wasnot originally expressed, the cell-surface expression was not observed.On the other hand, it was shown that the cell-surface expression of ILT1could be confirmed when ILT1 was coexpressed with the Fc receptorγ-chain (Nakajima H. et al., J. Immunology 162:5-8.1999). However, thereis no information about an immunogen for producing ILT7 antibodies.

For example, in the report, RBL cells into which ILT1 gene is introducedare used as immunogens to produce ILT1 antibodies. The present inventorstried to produce ILT7 antibodies using the combination of RBL cells withILT7 gene in the same manner as described. However, even if ILT7 wasforced to be expressed in RBL cells (P815), the cell-surface expressionof ILT7 was not observed, and therefore it could not be used as animmunogen.

The present inventors have conducted dedicated research in order toobtain the antibody capable of recognizing human ILT7. As a result, thepresent inventors found that the desired antibody could be produced byusing a specific transformed cell as an immunogen and completed thepresent invention. That is, the present invention relates to amonoclonal antibody which binds to the extracellular domain of humanILT7, and relates to a fragment comprising its antigen binding region.

In the present invention, human ILT7 can be defined as a naturalmolecule which is expressed in human IPCs or a molecule which isimmunologically equivalent to ILT7 which is expressed in human IPCs. Inthe present invention, the binding of antibodies to human ILT7 can beconfirmed, for example, as follows.

Confirmation Based on Responsiveness to Human Cells:

According to the findings of the present inventors, specific expressionof human ILT7 was observed in human IPCs. Originally, human ILT7 wasisolated as a gene whose expression is seen in Plasmacytoid dendriticcells (Blood. 2002 100; 3295-3303, Gene. 2004 Apr. 28; 331:159-64). Inaddition, it is also known that it can be used as a marker ofPlasmacytoid dendritic cells (WO03/12061). It is assumed thatPlasmacytoid dendritic cells and IPCs are mostly identical cellpopulations or their large portions are common. Therefore, there is nocontradiction between these reports and the findings of the presentinventors.

Considering such expression profile of human ILT7, first, the bindingactivity of IPCs or Plasmacytoid dendritic cells to at least a certainsubset is one of the important characteristics of the antibody whichbinds to human ILT in the present invention. Cell surface markersspecific to respective cell populations can be used to determine whethera certain cell is IPC or Plasmacytoid dendritic cell. For example,binding to the desired cells can be confirmed by double staining withthe antibody which binds to cell surface markers and the antibody whosebinding activity should be checked. That is, IPCs in the presentinvention comprises, for example, cells which express BDCA2.

Confirmation Based on Responsiveness to Transformed Cells ExpressingHuman ILT7 Gene:

The present inventors found that an immunological characteristic of ILT7expressed in human IPCs was reconstructed when expression of human ILT7gene was carried out under a specific condition. Therefore, theresponsiveness to human ILT7 can also be confirmed based on theresponsiveness of antibodies to cells into which a gene encoding ILT7 isartificially introduced. Namely, the present invention relates to amonoclonal antibody which comprises the amino acid sequence constitutingan extracellular domain as the extracellular domain and binds to amolecule coexpressed with the signal-transducing molecule or relates toa fragment comprising its antigen binding region. Here, theextracellular domain is composed of an amino acid sequence whichcorresponds to the 17th to 444th position of the N terminal amino acidsequence shown in SEQ ID NO: 2 (from 1 to 428 in SEQ ID NO: 2).

For example, the immunological characteristic of ILT7 expressed in humanIPCs is maintained in cells co-transfected with an expression vectorcomprising a DNA encoding human ILT7 and an expression vector comprisinga DNA encoding the signal-transducing molecule. Therefore, a transformedcell, which coexpresses human ILT7 and the signal-transducing molecule,is preferable to confirm the binding affinity of antibodies to theextracellular domain of human ILT7 in the present invention. In thepresent invention, it is desirable to use a cell, which is nottransformed as controls when the responsiveness of antibodies, isconfirmed by using the transformed cell. Further, it is also importantto confirm that the binding of antibodies is not detected using the samehost cell which expresses only the signal-transducing molecule as acontrol.

In the present invention, a molecule, which induces the expression ofhuman ILT7 on the cell surface, can be used as the signal-transducingmolecule for the coexpression. The signal-transducing molecule in thepresent invention can also be defined as a molecule which can impart theimmunological characteristic of natural human ILT7 to at least theextracellular domain of ILT7 molecule in a cell which expresses ILT7. Asused herein, the term “immunological characteristic” of natural humanILT7 means recognition by an antibody which binds to human IPCs.

Specifically, it is preferable to use Fc receptor γ-chain or DAP12 as asignal-transducing molecule. In the present invention, the Fc receptorγ-chain is particularly preferable as the signal-transducing molecule.The Fc receptor γ-chain is a molecule consisting of amino acid sequencesshown in SEQ ID NO: 16. The signal-transducing molecule may be afragment as long as human ILT7 to be coexpressed is localized at thecell surface. As long as human ILT7 to be coexpressed is localized atthe cell surface, the mutation or addition of the amino acid sequence ispermitted in the amino acid sequences shown in SEQ ID NO: 16. That is,the present invention provides methods for producing cells which producea monoclonal antibody which binds to the extracellular domain of humanILT7, comprising the following steps of:

(1) administering a cell which exogenously expresses a proteincomprising extracellular domain of human ILT7 and a molecule comprisingamino acid sequences described in SEQ ID NO: 16 to immune animals; and

(2) selecting an antibody producing cell which produces the antibodywhich binds to human ILT7 from antibody producing cells of the immuneanimals.

Subsequently, as the antibody which binds to human ILT7 in the presentinvention, it is preferable to use an antibody in which crossing withcell populations which are known to express ILT families other than ILT7is not observed. Specifically, as the antibody which binds to human ILT7in the present invention, it is preferable to use an antibody in whichthe binding to the cell populations which are known to express ILTfamilies other than ILT7 cannot be observed under the same condition asthe condition in which the binding to IPCs was confirmed. As alreadydescribed, for example, ILT2 and ILT3 are expressed in not only PDC butalso DC obtained from MDDC or CD34 positive cells (Gene. 2004 Apr. 28;331: 159-64). On the other hand, the expression ILT7 cannot be detecteddue to the differentiation of IPCs into dendritic cells. Therefore, theantibody cannot detect the binding to DCs obtained from MDDC or CD34positive cells under the condition in which the binding to IPCs can beconfirmed is comprised in the antibody which binds to human ILT7 in thepresent invention.

The following expression patterns as to other ILT family molecules havebeen reported (“The KIR Gene Cluster” Carrington, Mary and Norman, Paul.Bethesda (Md.): National Library of Medicine (US), NCBI; 2003, Gene.2004 Apr. 28; 331: 159-64). Therefore, an antibody which binds to humanIPCs or PDCs and whose binding to the following cells cannot beconfirmed is included in an antibody having specificity to ILT7:

ILT1; myeloid lineage cells (monocytes, DCs derived from monocytes,macrophages);

ILT2; PDCs, B cells, CD34 positive cells, DCs derived from CD34 positivecells, and DCs derived from monocytes;

ILT3; PDCs and DCs;

ILT5; monocytes, DCs derived from CD34 positive cells, and DCs derivedfrom monocytes; and

ILT8; monocyte lineage cells.

That is, the monoclonal antibody, which binds to the extracellulardomain of human ILT7 in the present invention preferably, comprises amonoclonal antibody which has the following immunologicalcharacteristics:

a) the monoclonal antibody binds to human IPCs; and

b) the binding of the monoclonal antibody to one or more cells selectedfrom the group consisting of monocytes, macrophages, B cells, CD34positive cells, and dendritic cells derived from these cells cannot beconfirmed under the condition for binding to human IPCs.

As the monoclonal antibody of the present invention, it is preferable touse an antibody in which the binding to monocytes, macrophages, B cells,CD34 positive cells, and dendritic cells derived from these cells cannotbe confirmed under the condition for binding, particularly to humanIPCs.

Alternatively, the monoclonal antibody, which binds to the extracellulardomain of human ILT7 in the present invention, preferably comprises amonoclonal antibody which has the following immunologicalcharacteristics:

c) the monoclonal antibody binds to the transformed cell which isco-transfected with an expression vector expressively carrying the DNAencoding human ILT7 and an expression vector expressively carrying theDNA encoding the signal-transducing molecule;

d) the binding to the host cell prior to transformation cannot beconfirmed under the condition for binding to the co-transfected cells asdescribed in c); or

the monoclonal antibody of the present invention comprises a monoclonalantibody which has the following immunological characteristics:

e) the binding to the host cell which expresses only thesignal-transducing molecule cannot be confirmed under the condition forbinding to the co-transfected cells as described in c).

In the present invention, the fact that anti-ILT7 monoclonal antibodydoes not intersect with the ILT family of other molecules can beconfirmed using cells in which each ILT family was forced to beexpressed. That is, for forced expression, cDNA encoding each ILT familyof amino acid sequences is introduced into an appropriate host cell. Theanti-ILT7 monoclonal antibody whose crossing should be confirmed is madeto contact with the obtained transformed cell. Then, it can be confirmedthat if the binding of the antibody to the cell, which expresses ILTfamily molecules other than ILT7, is not observed, the antibody is ableto immunologically distinguish between ILT7 and other ILT familymolecules. For example, in examples described below, the fact that theanti-ILT7 monoclonal antibody obtained by the present invention does notintersect with ILT1, ILT2, and ILT3 is confirmed. Therefore, apreferable example of the monoclonal antibody in the present inventionis the monoclonal antibody binding to ILT7 in which the binding to ILT1,ILT2, and ILT3 cannot be detected under the same condition.

Particularly, ILT2 and ILT3 are genes whose expression in IPCs has beenconfirmed (Ju et al. Gene 331, 159-164, 2004). However, these moleculesmay show expression profiles unique to each cell type depending on therespective differentiation levels in IPCs or conditions such as thestimulation with viruses or other cytokines. The use of an antibody,which is able to immunologically distinguish these ILT family moleculesfrom ILT7, allows for specifically detecting changes in the expressionof ILT7.

The binding of a monoclonal antibody whose binding activity should beconfirmed to various kinds of cells can be confirmed based on, forexample, the principle of flow cytometry. In order to confirm theresponsiveness of antibodies based on the principle of flow cytometry,it is advantageous to label antibodies with molecule or atomic groupwhich produces a detectable signal in advance. Generally, thefluorescent or luminescent labels are used.

Fluorescence-activated cell sorter (FACS) can be used to analyze thebinding of the fluorescent-labeled antibodies to cells based on theprinciple of flow cytometry. The use of FACS allows for efficientlyconfirming the binding of a plurality of antibodies to a plurality ofcells.

Specifically, for example, antibody A which has been previously found tobe able to identify IPCs and antibody B whose binding characteristics toIPCS should be analyzed are reacted with cell populations comprisingIPCs at the same time. Antibody A and antibody Bare labeled with afluorescence signal which is mutually distinguished by these antibodiesin advance. In the case where both signals are detected from the samecell populations, the binding of those antibodies to the same cellpopulations can be confirmed. In other words, it is found that bothantibodies A and B have the same binding characteristics. In the casewhere they bind to different cell populations, it is clear that bothantibodies have distinct binding characteristics.

A preferable example of the monoclonal antibody in the present inventionmay comprise a monoclonal antibody which is produced by hybridomaILT7#11 or ILT7#17. Hybridoma ILT7#11 and hybridoma ILT7#17 have beendeposited with National Institute of Advanced Industrial Science andTechnology, International Patent Organism Depositary under AccessionNos. FERM BP-10704 and FERM BP-10705 on Oct. 21, 2005.

The specified depository content is as follows:

(a) Appellation and address of depository institution Appellation:National Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary Address: AIST Tsukuba Central6, 1-1-1, Higashi, Tsukuba-shi, Ibaraki, Japan (zip code 305-9566)(b) Deposited date: Oct. 21, 2005(c) Accession number: FERM BP-10704 (hybridoma ILT7#11)(c) Accession number: FERM BP-10705 (hybridoma ILT7#17)

The monoclonal antibody of the present invention may be a fragmentcomprising its antigen binding region. For example, an antibody fragmentcomprising the antigen binding region which is obtained by enzymaticdigestion of IgG can be used as the antibody in the present invention.Specifically, antibody fragments such as Fab and F(ab′)₂ can be obtainedby digestion with papain or pepsin. It is well known that these antibodyfragments can be used as antibody molecules which have affinity forantibodies. Alternatively, antibodies constructed by geneticrecombination can also be used as long as satisfactory antigen-bindingactivity is maintained. Examples of the antibodies constructed bygenetic recombination comprise chimeric antibodies, CDR-transplantedantibodies, single chain Fvs, diabodies, linear antibodies, andpolyspecific antibodies formed of antibody fragments. It is commonknowledge that these antibodies can be given by using monoclonalantibodies or antibody producing cells which produce the antibodies.

The monoclonal antibody of the present invention can be obtained byusing a specific transformed cell as an immunogen. That is, the presentinvention relates to a method for producing cells which produce amonoclonal antibody which binds to the extracellular domain of humanILT7, comprising the following steps of:

(1) administering a cell which expresses an exogenous protein comprisingextracellular domain of human ILT7 and an exogenous molecule which isassociated with human ILT7 to immune animals; and

(2) selecting an antibody producing cell which produces the antibodywhich binds to human ILT7 from antibody producing cells of the immuneanimals.

The antibody producing cells thus obtained or the immortalized antibodyproducing cells are cultured and the desired monoclonal antibodies canbe recovered from the cultures. With reference to the method forimmortalizing antibody producing cells, various methods are known.

In the method for producing the monoclonal antibody of the presentinvention, usable examples of the molecule, which is associated withhuman ILT7 for producing a transformed cell to be used as an immunogen,comprise cell membrane proteins. Among them, a signal-transducingmolecule, which is localized in cell membranes, is preferable to use asa cell membrane protein in the present invention. The term“signal-transducing molecule” means a molecule which is associated withproteins and cells having receptor structures in the extracellulardomain and transmits the stimulation of binding of ligands to receptorsinto cells. Examples of the signal-transducing molecule comprise Fcreceptor γ-chain, DAP12, or the like. For example, Fc receptor γ-chainis preferable to use as a cell membrane protein in the presentinvention. Amino acid sequences of human DAP12 and Fc receptor γ-chainas well as a base sequence of cDNA, which encodes the sequences, arepublicly known. A base sequence of human Fc receptor γ-chain and anamino acid sequence which is encoded by the base sequence are shown inSEQ ID NOs: 15 and 16, respectively.

In the present invention, a transformed cell to be used as an immunogencan be obtained by preparing, for example, a cell expressively carryingthe following (a) and (b):

(a) an exogenous polynucleotide encoding an amino acid sequencecomprising an extracellular domain of human ILT7; and

(b) an exogenous polynucleotide encoding Fc receptor γ-chain.

In the present invention, an exogenous polynucleotide means apolynucleotide which is artificially introduced into a host cell. Whenhuman cells are used as cells, human genes are introduced into humancells. In such a combination, an artificially introduced polynucleotidemeans the exogenous polynucleotide. Therefore, ectopic expression ofhuman ILT7 or human Fc receptor γ-chain is comprised in expression ofthe exogenous polynucleotide.

As used herein, the term “extracellular domain of human ILT7” means theamino acid sequence from the 17th to 444th position of the amino acidsequence described in SEQ ID NO: 2 which corresponds to theextracellular domain of the amino acid sequence (from 1 to 428 in SEQ IDNO: 2). As an amino acid sequence comprising the extracellular domain ofhuman ILT7 in the present invention, it is preferable to use the aminoacid sequence which comprises each region, for example, starting fromthe N terminal, in order of the following:

[Signal sequence+extracellular domain+transmembrane domain+intracellularregion]

Alternatively, an amino acid sequence, which partially lacks anintracellular region as described below, is included in the amino acidsequence comprising the extracellular domain of human ILT7 in thepresent invention.

[Signal sequence+extracellular domain+transmembrane domain+a portion ofintracellular region]

Furthermore, a structure, which lacks an intracellular region asmentioned below, is included in the amino acid sequence comprising theextracellular domain of human ILT7 in the present invention. [Signalsequence+extracellular domain+transmembrane domain]

In the structure, regions other than the extracellular domain may beamino acid sequences which are selected from the amino acid sequenceshown in SEQ ID NO: 2, or may be combined with other amino acidsequences having homology with the regions. For example, the amino acidsequence constituting a signal sequence, a transmembrane domain, and anintracellular region may be an amino acid sequence of ILT familymolecules other than ILT7. Or, it may be combined with the amino acidsequence of ILT family in species other than human. Further, the aminoacid sequence, which constitutes regions other than the extracellulardomain, may comprise a mutation in the range capable of maintaining thefunction of each region. Alternatively, other regions may intervenebetween each region. For example, an epitope tag such as FLAG can alsobe inserted between the signal sequence and the extracellular domain.Particularly, the signal sequence is removed by processing during itstransfer to the cell membrane surface after being translated intoprotein. Therefore, arbitrary amino acid sequence, which induces transitof the translated protein to the cell membrane, can be used as thesignal sequence. More specifically, it is preferable to use the aminoacid sequence (SEQ ID NO: 2) of human ILT7 as the amino acid sequencecomprising the extracellular domain of human ILT7.

Therefore, in the present invention, an arbitrary base sequence whichencodes the amino acid sequence constituting the above-mentionedstructure [signal sequence+extracellular domain+transmembranedomain+intracellular region] can be used as the polynucleotide whichconstitutes the exogenous polynucleotide described in (a). For example,the amino acid sequence of SEQ ID NO: 2 is encoded by the base sequencedescribed in SEQ ID NO: 1.

In the present invention, an expression vector expressively carrying theabove-mentioned polynucleotides (a) and (b) may be introduced to anappropriate host cell in order to obtain a transformed cell to be usedas an immunogen. The Polynucleotides (a) and (b) can be carried on onevector or different vectors. When each polynucleotide is carried ondifferent vectors, the host cells are co-transfected with two kinds ofvectors.

Preferable examples of the host cell in the present invention comprisemammalian cells. Specific examples of the host cell comprise cellsderived from humans, monkeys, mice or rats. Particularly, the cellsderived from humans are preferable as host cells. For example, it ispreferable to use 293T cells derived from human as the host cell in thepresent invention. 293T cells can be obtained as ATCC CRL-11268. Inaddition, cells derived from immune animals can also be used as hostcells. When cells derived from immune animals are used as immunogens,little immunological response to host cells is given. For that reason,an antibody against the extracellular domain of exogenously expressedILT7 can be obtained efficiently. Therefore, for example, when mice areused as immune animals, cells derived from mice can also be used as hostcells.

The above-mentioned polynucleotides can be transformed into cells bycarrying them on a vector capable of inducing expression in host cells.Commercially available vectors, which can induce the expression inmammalian cells, may be used. Expression vectors such as pCMV-Script (R)Vector, PSG5 Vector (manufactured by Stratagene), pcDNA3.1 (manufacturedby Invitrogen) can be used for the present invention.

The transformed cells thus obtained are administered to immune animalstogether with additional components such as adjuvants, if necessary.Usable examples of the adjuvant include Freund's complete adjuvant, andthe like. In case of using mice as immune animals, the transformed cellscan be administered in the range of 10⁴ to 10⁹ cells, more specifically10⁴ to 10⁶ cells. Generally, multiple doses of immunogen are given atregular intervals until the antibody titer is elevated. For example, inthe case of a short-term immunization, the transformed cells areadministered at 2 to 4 day intervals, more specifically at intervals of3 days. After administering twice or three times, antibody producingcells can be recovered. Alternatively, they are administered once weeklyand antibody producing cells can also be recovered after administeringfive or six times.

In the present invention, the recovered antibody producing cells arecloned to give monoclonal antibodies. It is preferable that the antibodyproducing cells are immortalized for cloning. For example, the cellfusion method as typified by the hybridoma method or transformation byEpstein-Barr virus (EBV) can be used as the method of immortalization ofantibody producing cells.

As for antibody producing cells, one cell produces one kind of antibody.Therefore, the establishment of cell populations derived from one cell(i.e. cloning) allows for producing monoclonal antibodies. The hybridomamethod involves the process in which antibody producing cells are fusedwith an appropriate cell line, which is immortalized and then subjectedto cloning. The immortalized antibody producing cells can be cloned by atechnique such as limiting dilution method. It is known that there arelots of cell lines useful for the hybridoma method. These cell lines areexcellent in the immortalization efficiency of lymphocytic cells andhave various genetic markers which are needed to select the successfullyfused cells. Further, when the production of antibody producing cells isintended, a cell line lacking the ability to produce antibodies can alsobe used.

For example, mouse myelomas P3x63Ag8.653 (ATCC CRL-1580) and P3x63Ag8U.1(ATCCCRL-1597) are widely used as useful cell lines for the cell fusionmethod for mice or rats. In general, a hybridoma is produced by thefusion of homogeneous cells, while a monoclonal antibody can also beobtained from hetero hybridoma from a different species among closelyrelated species.

Specific protocols of the cell fusion have been publicly known. That is,antibody producing cells of immune animals are mixed with appropriatefusion partners to perform cell fusion. Usable examples of the antibodyproducing cell include splenic cells, lymphocyte cells collected fromthe lymph node, and peripheral blood B cells. As fusion partners,various cell lines described previously can be used. The polyethyleneglycol method and electric fusion method can be used for cell fusion.

Next, on the basis of selective markers of fused cells, the successfullyfused cells are selected. For example, when HAT sensitive cell line isused for cell fusion, the successfully fused cells are selected byselecting cells growing in HAT medium. Further, it is confirmed that theantibodies produced by the selected cells have the desiredresponsiveness.

Each hybridoma is screened based on the responsiveness of antibodies.That is, the hybridoma producing antibodies which bind to human ILT7 isselected by the method as described previously. Preferably, when theselected hybridoma is subcloned and then the production of the desiredantibody is finally confirmed, the confirmed antibody is selected as ahybridoma producing monoclonal antibody of the present invention.

Specifically, the desired hybridoma can be selected based on theresponsiveness to human cells or the responsiveness to the transformedcell which expresses human ILT7 gene. The antibodies, which bind tocells, can be detected based on the principle of immunoassay. Forexample, ELISA, which uses cells as antigens, can be utilized fordetection of the desired antibody. Specifically, a culture supernatantof hybridoma is made to contact with a support on which human IPC or thetransformed cell used as an immunogen. In the case where the culturesupernatant comprises the desired antibody, the antibody is trapped inthe cell immobilized on the support. Then, the solid phase is separatedfrom the culture supernatant, which is washed, if necessary. Thereafter,the antibody trapped in the solid phase can be detected. An antibody,which recognizes an antibody, can be used for the detection ofantibodies. For example, an antibody of mouse can be detected by ananti-mouse immunoglobulin antibody. The detection is easy if theantibody, which recognizes an antibody, is labeled. Usable examples ofthe label include enzymes, fluorescent dyes, luminescent dyes, and thelike.

On the other hand, particles and an inner wall of a microtiter plate canbe used as the support on which cells are immobilized. Cells can beimmobilized on particles made of plastic or the surface of a containerby physical adsorption. Usable examples of the support for immobilizingcells include beads made of polystyrene and reaction vessels.

In the selection of hybridomas, the production of the antibody againstnot ILT7 but the host cell of the transformed cell used as an immunogenmay be predicted. For example, as illustrated in Examples, in the casewhere a human cell is used as an immunogen and a mouse is used as animmune animal, the human cell is recognized as a foreign substance.Thus, the production of an antibody, which binds to the foreignsubstance, is predicted. In the present invention, it is intended toobtain an antibody capable of recognizing human ILT7. Therefore, it isnot necessary to obtain an antibody which recognizes human cell antigensother than human ILT7. In order to remove hybridomas, which produce suchan antibody in screening, undesired antibodies, can be absorbed prior tothe confirmation of the antibody responsiveness.

Undesired antibodies can be absorbed by an antigen to which an antibodypresumed to exist binds. Specifically, for example, an antibody againsthuman cell antigens other than human ILT7 can be absorbed by a cellwhich cannot detect the expression of human ILT7. In the presentinvention, it is preferable to use the host cell used for the immunogenas an antigen for absorbing the undesired antibodies. Alternatively, ahost cell which does not express the extracellular domain of human ILT7,but expresses molecule which associates with ILT7 can be used as theantigen for absorbing the antibodies.

As for the monoclonal antibody whose binding activity to antigen isconfirmed, its actual effect on the IPC activity is confirmed, ifnecessary. The effect on IPC can be confirmed by methods such as themethods described below.

As for the monoclonal antibody of the present invention, a hybridomaproducing the monoclonal antibody is cultured and the monoclonalantibody of the present invention is recovered from the resultingculture. The hybridoma can be cultured in vitro or in vivo. In the caseof in vitro, the hybridoma can be cultured by using a known culturemedium such as RPMI1640. The immunoglobulin secreted by the hybridoma isaccumulated in the culture supernatant. Therefore, the monoclonalantibody of the present invention can be obtained by collecting theculture supernatant and purifying it, if necessary. It is easier topurify the immunoglobulin when serum is not added to the culture medium.However, for the purpose of more rapid proliferation of the hybridomaand facilitation of antibody production, 10% fetal bovine serum can alsobe added to the culture medium.

The hybridoma can also be cultured in vivo. Specifically, theintraperitoneal cultivation of the hybridoma can be made by inoculatingthe hybridoma into the abdominal cavity of nude mice. Monoclonalantibodies are accumulated in the ascites. Therefore, if the ascites isobtained and purified as needed, the required monoclonal antibody can beproduced. The obtained monoclonal antibodies can be appropriatelymodified or processed in accordance with the intended use.

The monoclonal antibody of the present invention can be expressed byobtaining cDNA which encodes the antigen binding region of antibody fromthe hybridoma and inserting into an appropriate expression vector. Thetechnique, in which cDNA which encodes a variable region of antibody isobtained and then it is expressed in an appropriate host cell, is known.In addition, the method in which a chimeric antibody is made by ligatinga variable region comprising the antigen binding region into a constantregion is also known.

Preferable examples of the monoclonal antibody in the present inventioncomprise monoclonal antibodies produced by hybridoma #11 (Accessionnumber: FERM BP-10704), hybridoma #17 (Accession number: FERM BP-10705),or hybridoma #37. Amino acid sequences which constitute variable regionsof these monoclonal antibodies as well as base sequences of cDNAencoding thereof are described below. Therefore, for example, chimericantibodies to be obtained by conjugating these variable regions toconstant regions of other immunoglobulins are preferable in the presentinvention. In amino acid sequences described in the Sequence Listing,the amino acid sequence from 1 to C terminus constitutes a matureprotein. That is, the consecutive amino acid sequence from 1 to Cterminus for each amino acid sequence is a mature sequence of each aminoacid sequence. On the other hand, the amino acid sequence represented bya numerical value from N terminus to −1 is a signal sequence.

Heavy chain variable region Light chain variable region #11 SEQ ID NO:38 SEQ ID NO: 40 (base sequence) (base sequence) SEQ ID NO: 39 SEQ IDNO: 41 (amino acid sequence) (amino acid sequence) #17 SEQ ID NO: 42 SEQID NO: 44 (base sequence) (base sequence) SEQ ID NO: 43 SEQ ID NO: 45(amino acid sequence) (amino acid sequence) #37 SEQ ID NO: 46 SEQ ID NO:48 (base sequence) (base sequence) SEQ ID NO: 47 SEQ ID NO: 49 (aminoacid sequence) (amino acid sequence)

For example, a mouse (variable region)-human (constant region) chimericantibody can be made by ligating these variable region genes into ahuman IgG1 heavy chain constant region and a gene encoding human Igkappa light chain constant region, respectively. Amino acid sequences ofsuch a chimeric antibody and base sequences encoding thereof arerespectively described below. Chimeric antibodies specified by thesesequences show the construction of a preferred embodiment of anti-ILT7monoclonal antibody in the present invention. In the following aminoacid sequences of chimeric antibodies, the amino acid sequence from Nterminus to −1 corresponds to the signal sequence and the amino acidsequence from 1 to C terminus corresponds to the mature protein. Thatis, a chimeric antibody comprised of heavy and light chains, whichconsist of the amino acid sequence from 1 to C terminus for each aminoacid sequence, is preferable in the present invention.

Heavy chain Light chain #11 SEQ ID NO: 50 SEQ ID NO: 52 (base sequence)(base sequence) SEQ ID NO: 51 SEQ ID NO: 53 (amino acid sequence) (aminoacid sequence) #17 SEQ ID NO: 54 SEQ ID NO: 56 (base sequence) (basesequence) SEQ ID NO: 55 SEQ ID NO: 57 (amino acid sequence) (amino acidsequence)

Further, the antigen-binding activity of monoclonal antibody can also begrafted to other immunoglobulins. The variable region of immunoglobulinis comprised of a complementarity-determining region (CDR) and a frameregion. The antigen-binding property of each immunoglobulin isdetermined by CDR and the frame maintains the structure of antigenbinding region. The amino acid sequence of CDR is extremely rich indiversity, while the amino acid sequence of the portion of the frame ishighly conserved. It is known that the amino acid sequence constitutingCDR is incorporated into the frame region of other immunoglobulinmolecules, which allows for grafting of the antigen-binding activity.The method in which the antigen-binding property of differentimmunoglobulins is grafted to human immunoglobulin by using this processhas been established. As used herein, the term “antigen binding region”can comprise the CDR which is grafted to the frame. Therefore, the term“fragment comprising the antigen binding region” of a certain monoclonalantibody” comprises a fragment of human immunoglobulin comprising thevariable region to which CDR of the monoclonal antibody is grafted. Forexample, each of the amino acid sequences of the above-mentionedvariable regions comprises the following amino acid sequences (SEQ IDNOs) as CDRs.

CDR1 CDR2 CDR3 #11 heavy SDYAWN YISYSGSTSYNPSLKSR SPPYYAMDY chain (58)(59) (60) #11 light KASQDVGTAVA WASTRHT QQYSSYPLT chain (61) (62) (63)#17 heavy SYWIH RIYPGTGSTYYNEKFKG YPTYDWYFDV chain (64) (65) (66)#17 light RASQSISNYLH YASQSIS QQSNSWPLT chain (67) (68) (69) #37 heavySDYAWN YISYSGSTSYNPSLKSR ALPLPWFAY chain (70) (71) (72) #37 lightKASQDVGTAVA WASTRHT QQYSSYPYT chain (73) (74) (75)

Based on the information of the base sequence which encodes theabove-mentioned amino acid sequences and the information of the basesequence which encodes the frame (FR) of human immunoglobulin, a primercan be designed and cDNA having a base sequence obtained by conjugatingboth of the base sequences can be amplified. The operation is repeatedfor each frame and a variable region in which CDR1, CDR2, and CDR3 ofmice are connected by human FR can be constructed. Further, when thebase sequence, which encodes a constant region of human immunoglobulin,is conjugated as needed, a humanized antibody with the constant regioncan be obtained.

As the chimeric antibody comprising the above-mentioned variable regionsor a humanized antibody to which CDR constituting a variable region isgrafted, an antibody with a constant region derived from IgG or IgM iscomprised in a preferable antibody in the present invention. The presentinventors confirmed that the monoclonal antibody against ILT7 showed CDCaction on ILT7 expressing cells. Therefore, the antibody having aconstant region derived from IgG or IgM exhibits cytotoxicity againstILT7 expressing cells due to CDC effect. Such antibodies are useful ininhibiting the number of ILT7 expressing cells such as IPCs.

The chimeric antibody capable of recognizing ILT7 or humanized antibody,which is provided by the present invention, can be produced by geneticengineering using polynucleotides encoding these antibodies. Forexample, a polynucleotide which is the base sequence described in thefollowing SEQ ID NOs and encodes the amino acid sequence constituting amature protein for each amino acid sequence can be used as apolynucleotide encoding the variable region #11 or #17. The consecutiveamino acid sequence from 1 to C terminus for each amino acid sequencecorresponds to a mature protein. In the case where each mature proteinis expressed as a separate protein, it is preferable to place thesecretion signal at the N terminus of each amino acid sequence. Forexample, in the amino acid sequences shown in these SEQ ID NOs, theamino acid sequence from N terminus to −1 can be used as a signalsequence when such proteins are expressed in animal cells.Alternatively, these variable regions can be secreted as mature proteinsby using an arbitrary signal sequence which enables the secretion ofimmunoglobulin.

#11 SEQ ID NO: 50 (base sequence) SEQ ID NO: 52 (base sequence) #17 SEQID NO: 54 (base sequence) SEQ ID NO: 56 (base sequence)

In the same manner as described above, as for the polynucleotideencoding the humanized antibody, a polynucleotide which expresses thehumanized antibody can be made by using the base sequence which encodesa protein having the signal sequence to be added to the N terminus. Whenheavy and light chains are carried on separate vectors, both vectors areco-transfected into the same host cell. The heavy and light chainsexpressed from each vector are used to construct an immunoglobulinmolecule with both chains. Or, a polynucleotide encoding a heavy chainand a polynucleotide encoding a light chain can also be carried on thesame vector. The host cell into which a vector carrying bothpolynucleotides is co-transfected expresses heavy and light chains andproduces an immunoglobulin having both chains.

These polynucleotides can be expressed as antibodies using a host-vectorsystem capable of expressing an antibody gene. Furthermore, in the casewhere they are expressed as a single protein molecule by connecting aheavy chain variable region with a light chain variable region, a signalsequence can be placed at the N terminus of the protein molecule. Aknown example of such an antibody molecule includes scFv molecule inwhich a heavy chain variable region and a light chain variable regionare connected by a linker.

Each of the monoclonal antibodies thus produced is comprised in themonoclonal antibody of the present invention. In other words, amonoclonal antibody which consists of an immunoglobulin comprising theantigen binding region encoded by a polynucleotide derived from cDNAencoding the antigen binding region of the above-mentioned monoclonalantibodies is comprised in the monoclonal antibody in the presentinvention.

As described previously, RBL cells in which ILT1 gene was forced to beexpressed could be used as an immunogen for obtaining ILT1 antibodies.However, the expression of ILT7 on the surface of RBL cells (P815) couldnot be confirmed and thus it could not be used as the immunogen. Thepresent inventors found out that the expression of human ILT7 on thecell surface could be induced by the coexpression of human ILT7 andother cell membrane proteins which associate with human ILT7. Then, thepresent inventors found that the antibody, which binds to human IPCs,can be obtained by using the transformed cell whose expression is thusinduced as an immunogen and completed the present invention.

That is, the present invention provides the immunogen for producing theantibody which binds to the extracellular domain of human ILT7, andcomprises animal cells in which (a) a polynucleotide which encodes theamino acid sequence comprising the extracellular domain of human ILT7;and (b) a polynucleotide which encodes Fc receptor γ-chain aremaintained so as to be exogenously expressed or cell membrane fractionsthereof.

Six years or more have already passed since the structure of human ILT7was found in 1998. However, the antibody capable of specificallyrecognizing ILT7 has still not been obtained. The antibody capable ofrecognizing human ILT7 was provided by using the immunogen of thepresent invention for the first time. That is, the present inventionprovided the antibody capable of recognizing human ILT7 which can beobtained by the following steps of:

(1) administering a cell which exogenously expresses a proteincomprising extracellular domain of human ILT7 and a molecule which isassociated with human ILT7 to immune animals;

(2) selecting an antibody producing cell which produces the antibodywhich binds to human ILT7 from antibody producing cells of the immuneanimals; and

(3) culturing the antibody producing cells selected by step (2) andrecovering an antibody capable of recognizing human ILT7 from thecultures.

It is found that human ILT7 is specifically expressed in human IPC. Inthe analysis of gene expression by SAGE which was performed by thepresent inventors, the specific expression of human ILT7 in human IPCwas also confirmed. However, in the past reports, the expression levelsof ILT7 of both cases were analyzed based on mRNA. Since the antibodycapable of detecting human ILT7 was not provided, the expression stateof protein was not analyzed conventionally. The analysis of human ILT7protein was realized by the provision of the antibody which binds to theextracellular domain of human ILT7 in the present invention.

The present inventors actually confirmed that the monoclonal antibodywhich binds to the extracellular domain of human ILT7 based on thepresent invention specifically detected human IPCs. That is, the presentinvention relates to a method for detecting interferon producing cellswhich comprise the steps of: contacting a monoclonal antibody whichbinds to the extracellular domain of human ILT7 or a fragment comprisingthe antigen binding region with a test cell; and detecting themonoclonal antibody which is bound to cells or a fragment comprising itsantigen binding region.

The detection of human ILT7 based on the present invention allows fordetermining whether a certain cell is IPC. That is, the presentinvention provides a method for identifying IPCs using human ILT7 as anindicator. Or, human IPCs can be separated by separating the cells inwhich human ILT7 was detected based on the present invention. That is,the present invention provides a method for separating IPCs using humanILT7 as an indicator.

Based on the analysis by human ILT7 antibody, it was confirmed that theexpression level of ILT7 in IPCs whose differentiation was induced byCpG, and the like was reduced. That is, the IPCs before theirdifferentiation is induced can be specifically detected by using ILT7 asan indicator. In other words, the monoclonal antibody of the presentinvention is useful, particularly in detecting IPCs before theirdifferentiation into dendritic cells. As used herein, the term “IPCsbefore their differentiation” can be defined as cell populations whichmaintain the capacity to produce interferon.

In the present invention, the monoclonal antibody which binds to theextracellular domain of human ILT7 or the fragment comprising itsantigen binding region can be labeled in advance. For example,antibodies can be easily detected by labeling with luminescent dyes orfluorescent dyes. More specifically, the fluorescent-dye labeledantibody is made to contact with a cell population which may compriseIPCs and then cells to which the antibody of the present invention boundcan be detected by using the fluorescent dye as an indicator. Further,IPCs can be separated by separating the cells in which the fluorescentdye is detected. A series of the steps can be easily performed based onthe principle of FACS.

Alternatively, the antibody of the present invention can be bound to asolid phase support such as magnetic particles in advance. The antibodybound to the solid phase support recognizes human ILT7 and then IPCs aretrapped in the solid phase support. As a result, IPCs can be detected orseparated.

The antibody necessary for the method for detecting IPCs based on thepresent invention can be provided as a reagent for detecting IPCs. Thatis, the present invention provides a reagent for detecting interferonproducing cells, comprising the monoclonal antibody which binds to theextracellular domain of human ILT7 or the fragment comprising itsantigen binding region. The reagent for detecting IPCs of the presentinvention can be used in combination with a positive control or anegative control in addition to antibodies. For example, the transformedcells which express the extracellular domain of human ILT7 and are usedfor the immunogen as well as the IPCs obtained from human can be used asthe positive controls. Usually, only a few human IPCs can be obtainedfrom the peripheral blood. Therefore, it is preferable to use,particularly a transformed cell as the positive control in the reagentof the present invention. On the other hand, an arbitrary cell, whichdoes not express human ILT7, can be used as the negative control.

That is, the present invention provides a kit for detecting human IPCswhich comprises:

(a) the monoclonal antibody which binds to the extracellular domain ofhuman ILT7 or the fragment comprising its antigen binding region; and

(b) the cell which expresses an exogenous protein comprisingextracellular domain of human ILT7 and an exogenous molecule which isassociated with human ILT7.

The present inventors analyzed the effect of the antibody which binds tothe extracellular domain of human ILT7 on IPCs. As a result, it isconfirmed that the antibody, which binds to the extracellular domain ofhuman ILT7, inhibits the activity of IPCs. That is, the presentinvention relates to a method for inhibiting the activity of interferonproducing cells, comprising a step of contacting any of the followingcomponents with interferon producing cells:

(a) a monoclonal antibody which binds to human ILT7 and inhibits theactivity of interferon producing cells or a fragment comprising itsantigen binding region; and

(b) an immunoglobulin to which a complementarity-determining region ofthe monoclonal antibody described in (a) is grafted or a fragmentcomprising its antigen binding region.

Or, the present invention relates to a method for inhibiting theactivity of interferon producing cells in living organisms, comprising astep of administering any of the following components to the livingorganisms:

(a) the monoclonal antibody which binds to human ILT7 and inhibits theactivity of interferon producing cells or a fragment comprising itsantigen binding region;

(b) a fragment comprising the immunoglobulin to which acomplementarity-determining region of the monoclonal antibody describedin (a) is grafted or a fragment comprising its antigen binding region;and

(c) a polynucleotide which encodes the components described in (a) or(b).

As used herein, the term “Interferon Producing cells (IPCs)” means cellswhich have the ability to produce IFN and express ILT7 on the cellsurface. Hereinafter, unless otherwise noted, the term “IPCs”encompasses not only cells which are precursor cells of dendritic cellsbut also the cells which have the ability to produce IFN and expressILT7 on the cell surface. Methods for identifying such IPCs are commonlyknown. IPCs can be distinguished from other blood cells using some cellsurface markers as indicators. Specifically, a profile of cell surfacemarkers of human IPCs is described below (Shortman, K. and Liu, Y J.Nature Reviews 2: 151-161, 2002). In recent years, a certain report hasalso suggested that BDCA-2 positive cell is defined as IPC (Dzionek, A.et al. J. Immunol. 165: 6037-6046, 2000).

[Profile of Cell Surface Antigens of Human IPCs] CD4 Positive, CD123Positive, Lineage (CD3, CD14, CD16, CD19, CD20, CD56) Negative, andCD11c Negative

Therefore, it can also be said that IPCs are cells which have theexpression profile of these known markers and have the ability toproduce IFN. Further, cells in living organisms with the ability toproduce IFN are comprised in IPCs, even if the cells are a cellpopulation with profiles different from the expression pattern of theexpression profile of these markers. Further, examples of thecharacteristics, which are commonly seen in human IPCs, are as follows:

[Morphological Characteristic of Cells]

-   -   Similar to plasma cells    -   Round cells with a smooth cell surface    -   The nucleus is relatively large

[Functional Characteristic of Cells]

-   -   During virus infection, a large amount of Type-1 interferons are        produced in a short period of time.    -   Differentiated into dendritic cells after virus infection.

As used herein, the term “inhibition of the activity of IPCs” means theinhibition of at least one of the functions of IPCs. Examples of thefunction of IPCs include the production of IFN and the cell survival.The cell survival can also be translated into the number of cells.Therefore, in the case of inhibiting both or either of these functions,it is said that the activity of IPCs is inhibited. It is found that type1 IFN produced by IPCs leads to various diseases. Therefore, theinhibition of the number of IPCs and IFN production is useful for amedical treatment strategy of those diseases.

For example, the relationship between the pathological condition ofautoimmune diseases and IFNα has been pointed out. Most of the IFNα isproduced by IPCs. Therefore, pathological conditions caused by IFNα canbe alleviated by inhibiting the production of IFNα. As used herein, theterm “inhibition of IFN production by IPCs” means the inhibition of theproduction of at least one of the IFN produced by IPCs. Preferable IFNin the present invention is the type 1 IFN. Among them, IFNα isimportant.

That is, the present invention relates to an inhibitor of the productionof IFN which comprises an antibody which binds to the extracellulardomain of ILT7 as an active ingredient. Or, the present inventionprovides a method for inhibiting the production of IFN comprising a stepof administering the antibody which binds to the extracellular domain ofILT7. Further, the present invention relates to the use of the antibodywhich binds to the extracellular domain of ILT7 in the production of amedicinal composition for inhibiting the production of IFN.

Cells in which a large amount of IFN is produced by a small number ofcells are included in IPCs. For example, precursor cells of dendriticcells stimulated by viruses and the like produce most of the IFNproduced by the living body. The inhibition of the number of IPCs whichproduce a lot of IFN results in suppressing the IFN production.Therefore, pathological conditions caused by IFNα can be reduced byinhibiting the number of IPCs. It was confirmed that anti-ILT7monoclonal antibody bound to ILT7 expressing cells and then the effectof cytotoxicity was given by Complement Dependent Cytotoxicity (CDC) ina preferable embodiment of the present invention. CDC effect is one ofthe important mechanisms of antibody drug. The anti-ILT7 monoclonalantibody of the present invention also has potent cytotoxicity againstILT7 expressing cells such as IPCs due to CDC effect thereof. That is,as for the anti-ILT7 monoclonal antibody, the IFN production inhibitingeffect can be expected by cytotoxicity against IPCs, in addition to theinhibition mechanism of IFN production in a preferable embodiment.

The antibody, which recognizes the extracellular domain of human ILT7 tobe used for the present invention, can be obtained based on the methoddescribed previously. The antibody in the present invention may be ofany class. Organism species from which the antibody is derived are notlimited, either. Further, a fragment comprising the antigen bindingregion of antibody can be used as an antibody. For example, an antibodyfragment comprising the antigen binding region which is obtained byenzymatic digestion of IgG can be used as the antibody in the presentinvention. Specifically, antibody fragments such as Fab and F(ab′)₂ canbe obtained by digestion with papain or pepsin. It is well known thatthese antibody fragments can be used as antibody molecules which haveaffinity for antibodies. Alternatively, antibodies constructed bygenetic recombination can also be used as long as satisfactoryantigen-binding activity is maintained. Examples of the antibodiesconstructed by genetic recombination include chimeric antibodies,CDR-transplanted antibodies, single chain Fvs, diabodies, linearantibodies, and polyspecific antibodies formed of antibody fragments. Itis common knowledge that these antibodies can be given by usingmonoclonal antibodies.

In the present invention, antibodies can be modified, if necessary.According to the present invention, the antibody, which recognizes theextracellular domain of human ILT7, has an inhibiting effect on theactivity of IPCs. That is, it is contemplated that the antibody itselfhas cytotoxicity against IPCs. Subclasses of antibodies which exhibitpotent effector activity are known. Alternatively, the inhibiting effecton the IPC activity can be further enhanced by modifying antibodies witha cytotoxic agent. Examples of the cytotoxic agent are described below.

Toxins: Pseudomonas Endotoxin (PE), diphtheria toxin, ricinRadioisotopes: Tc^(99m), Sr⁸⁹, I¹³¹, Y⁹⁰Anticancer agents: calicheamicin, mitomycin, paclitaxel

Toxins consisting of proteins can be conjugated to antibodies or theirfragments with a bifunctional reagent. Alternatively, a gene encodingtoxin is connected to a gene encoding antibody and fusion proteins ofboth genes can also be obtained. The method for conjugating antibodieswith radioisotopes is also known. For example, the method for labelingantibodies with radioisotopes using a chelating agent is known.Furthermore, the anticancer agents can be conjugated to antibodies usingsugar chains or the bifunctional reagent.

The present inventors have confirmed a phenomenon in which a monoclonalantibody which is bound to ILT7 expressed on a cell membrane isincorporated into cells after binding (internalization). Therefore, thecytotoxic agents can be delivered into cells by contacting antibodiesconjugated with these cytotoxic agents of the present invention withILT7 expressing cells. That is, the present invention provides an activeinhibitor of ILT7 expressing cells which comprises anti-ILT7 monoclonalantibody to which the cytotoxic agent is conjugated as an activeingredient. Or, the present invention relates to the use of anti-ILT7monoclonal antibody to which the cytotoxic agent is conjugated in theproduction of the active inhibitor of ILT7 expressing cells. Further,the present invention provides a method for inhibiting the activity ofILT7 expressing cells comprising a step of administering anti-ILT7monoclonal antibody to which the cytotoxic agent is conjugated.

In the present invention, an antibody whose structure is artificiallymodified can also be used as an active ingredient. For example, variousmodification methods are known in order to improve the cytotoxicity andstability of antibodies. Specifically, an immunoglobulin in which sugarchains of heavy chains are modified is known (Shinkawa, T. et al. J.Biol. Chem. 278:3466-3473. 2003). Antibody Dependent Cell-mediatedCytotoxicity (ADCC) activity of immunoglobulin was enhanced by themodification of sugar chains. Or, an immunoglobulin in which the aminoacid sequence of Fc region is modified is also known. That is, ADCCactivity was enhanced by artificially increasing the binding activity ofimmunoglobulin to Fc receptor (Shield, R L. et al. J. Biol. Chem. 276;6591-6604, 2001).

IgG, which is bound to Fc receptor, is incorporated in cells once. Then,IgG binds to Fc receptor which is expressed in endosome and it isreleased into blood again. This phenomenon has been revealed. IgG with ahigh binding activity with Fc receptor has a better chance of beingreleased into blood again after its incorporation into cells. As aresult, the retention time of IgG in blood is extended (Hinton, P R. etal. J Biol Chem. 279: 6213-6216. 2004). In addition to this, it is saidthat modification of amino acid sequence of Fc region causes a change ofcomplement dependent cytotoxicity (CDC) activity. These modifiedantibodies can be used as the antibody in the present invention.

When the antibody, which binds to the extracellular domain of humanILT7, is contacted to IPCs, the activity of IPCs is inhibited.Therefore, these antibodies can be used for an inhibitor or method forinhibiting the activity of IPCs. That is, the present invention providesan active inhibitor of IPCs which comprises at least one componentselected from the group consisting of the following (a) to (c) as anactive ingredient. Or, the present invention relates to a method forinhibiting the activity of IPCs comprising a step of administering atleast one component selected from the group consisting of the following(a) to (c). Further, the present invention relates to the use of atleast one component selected from the group consisting of the following(a) to (c) in the production of active inhibitor of IPCs:

(a) the monoclonal antibody which binds to human ILT7 or a fragmentcomprising its antigen binding region;

(b) the immunoglobulin to which a complementarity-determining region ofthe antibody described in (a) is grafted or a fragment comprising itsantigen binding region; and

(c) a polynucleotide which encodes components described in (a) or (b).

In the present invention, the monoclonal antibody, which recognizes theextracellular domain of human ILT7, can be used as the monoclonalantibody which inhibits the activity of IPCs. In the present invention,one or more monoclonal antibodies can be used. For example, one or moremonoclonal antibodies, which recognize the extracellular domain of humanILT7, are blended to use in the present invention.

It can be confirmed that antibodies have an inhibiting effect on IFNproduction by IPCs in the manner as described below. IPCs produce alarge amount of IFN due to virus stimulation. Antibodies are given toIPCs before, after, or at the same time as the stimulation of IPCs withviruses. The capacity to produce IFN each for the resulting IPCs iscompared to that of each control to which antibodies are not given. Theability to produce IFN can be evaluated by measuring IFNα or IFNβcontained in culture supernatant of IPCs. As a result of the comparison,it can be confirmed that the tested antibodies are effective ininhibiting the ability to produce IFN when the amount of IFN in thesupernatant is significantly decreased by the addition of antibodies.These methods for measuring IFN are known. IPCs produce most of the IFNin the living body. Therefore, IFN producing state in the living bodycan be regulated by inhibiting the ability to produce IFN of IPCs.

In the present invention, the activity of IPCs encompasses themaintenance of the number of IPCs. Therefore, the inhibition of theactivity of IPCs in the present invention comprises the inhibition ofthe number of IPCs. When it is confirmed that the number of IPCs isinhibited under the presence of antibodies, it can be found that theantibodies are inhibiting the activity of IPCs. As with IFN production,an inert immunoglobulin derived from the same animal species as theantibody whose activity should be confirmed can be used as a comparativecontrol. The number of IPCs can be quantitatively compared by countingcells. The number of cells can be counted with FACS or a microscope.

Further, it is said that IPCs are differentiated into cells which induceTh2 referred to as dendritic cell 2 (DC2) as a result of infection withvirus or the like. If IFN production of IPCs by virus stimulation can beinhibited, their differentiation into Th2 may also be inhibited.Therefore, it can be expected that the monoclonal antibody of thepresent invention, which inhibits IFN production, may also have atherapeutic effect on various allergic diseases.

When the antibody, which recognizes the extracellular domain of humanILT7, is administered to a host different from organism species fromwhich the antibody is derived, it is desirable to process the antibodyinto a shape which is hard to be recognized as a foreign substance bythe host. For example, immunoglobulin cannot be easily recognized as theforeign substance by processing the antibody into the followingmolecules. The technique for processing immunoglobulin molecules asdescribed below is known. Fragment comprising the antigen binding regionwhich lacks a constant region (Monoclonal Antibodies: Principles andPractice, third edition, Academic Press Limited. 1995; AntibodyEngineering, A Practical Approach, IRL PRESS, 1996)

Chimeric antibody composed of the antigen binding region of monoclonalantibody and the constant region of immunoglobulin of the host (“GeneExpression Experiment Manual”, Isao Ishida, Tamie Ando, eds., Kodansha,1994)

CDR-substituted antibody in which complementarity-determining region(CDR) of immunoglobulin of the host is substituted to CDR of monoclonalantibody (“Gene Expression Experiment Manual”, Isao Ishida, Tamie Ando,eds., Kodansha, 1994)

Alternatively, a human antibody can be obtained by using non-humananimals into which a human antibody gene is incorporated as immuneanimals, while the non-human animals are used. For example, transgenicmice with human antibody genes have been put to practical use in orderto produce human antibodies as immune animals (Ishida et al., Cloningand Stem Cells, 4:85-95, 2002). The use of such animals allows forobtaining the human antibody which recognizes ILT7 using immunogens asdescribed previously. It is preferable to administer 16 human antibodyto humans.

Alternatively, a human immunoglobulin variable region gene can also beobtained by a phage display method (McCafferty J. et al., Nature348:552-554, 1990; Kretzschmar T et. al., Curr Opin Biotechnol. 2002December; 13(6): 598-602). In the phage display method, a gene encodinghuman immunoglobulin variable region is incorporated into a phage gene.A phage library can also be produced by using various immunoglobulingenes as sauces. A phage expresses a variable region as a fusion proteinof the protein composed of the phage. The variable region expressed bythe phage on the phage surface maintains the binding activity with anantigen. Therefore, phages, which bind to antigens or cells in whichantigens are expressed, are selected, thereby allowing for screening aphage in which a variable region having the desired binding activity isexpressed from the phage library. Further, a gene encoding a variableregion, which has the desired binding activity, is retained in the phageparticles thus selected. That is, in the phage display method, a geneencoding a variable region with the desired binding activity can beobtained by using the binding activity of the variable region as anindicator.

In the active inhibitor of IPCs or the method for inhibiting theactivity of IPCs in the present invention, the antibody which recognizesthe extracellular domain of human ILT7 or the antibody fragment whichcomprises at least the antigen binding region of the antibody can beadministered as a protein or a polynucleotide encoding the protein. Inadministration of polynucleotides, it is desirable to use a vector inwhich a polynucleotide encoding the desired protein is placed under thecontrol of an appropriate promoter so as to express the desired protein.An enhancer and a terminator can also be placed in the vector. A vectorwhich carries a gene of heavy and light chains which constitutes animmunoglobulin and is able to express an immunoglobulin molecule isknown.

The vector capable of expressing the immunoglobulin can be administeredby introducing into cells. In the administration to living organisms, avector, which can be infected with cells by administering to the livingorganisms, can be administered directly. Once lymphocytes are separatedfrom the living organisms, then the vector is introduced into thelymphocytes, which can be returned to the living organisms again (exvivo).

In the active inhibitor of IPCs or the method for inhibiting theactivity of IPCs based on the present invention, as for the amount ofmonoclonal antibody to be administered to the living organisms,immunoglobulin is administered usually in the range of 0.5 mg to 100 mg,for example, 1 mg to 50 mg, preferably 2 mg to 10 mg per kg of bodyweight. Intervals of administration of the antibody to living organismscan be properly adjusted so as to maintain an effective concentration ofimmunoglobulin in the living organisms during the period of treatment.Specifically, for example, the antibody can be administered at intervalsof 1 to 2 weeks. The administration route is optional. Those skilled inthe art can properly select an effective administration route intreatments. Specific examples thereof include oral or parenteraladministration. Antibodies are administered systemically or topicallyfor example, by intravenous injection, intramuscular injection,intraperitoneal injection, subcutaneous injection, or the like. Examplesof an appropriate formulation for parenteral administration in thepresent invention include injectable solutions, suppositories, andsprays. When the antibody is given to cells, immunoglobulin is added toculture medium usually in the range of 1 μg/ML, preferably 10 μg/ML,more preferably 50 μg/ML, further preferably 0.5 mg/ML.

In the active inhibitor of IPCs or method for inhibiting the activity ofIPCs of the present invention, the monoclonal antibody can beadministered to living organisms by optional methods. Usually, themonoclonal antibody is blended with a pharmaceutically acceptablesupport. If necessary, the monoclonal antibody can be blended withadditive agents such as thickeners, stabilizers, preservatives, andsolubilizers. Examples of such a support or additive agent includelactose, citric acid, stearic acid, magnesium stearate, sucrose, starch,talc, gelatin, agar, vegetable oil, and ethylene glycol. The term“pharmaceutically acceptable” means approved by a regulatory agency ineach government or listed in the Pharmacopeia in each country or othergenerally recognized pharmacopeia for use in animals, in mammalians, andmore particularly, in humans. The active inhibitor of IPCs of thepresent invention can also be provided in the form of single or multipledoses of lyophilized powders or tablets. Further, the lyophilizedpowders or tablets can be used in combination with sterilized water forinjection, physiological salt solution or buffer solution for dissolvingthe compositions so as to be a desired concentration beforeadministration.

Further, when the monoclonal antibody is administered as a vector, whichexpresses immunoglobulin, heavy and light chains are co-transfected toanother plasmid and each plasmid can be administered in the range of 0.1to 10 mg, for example, 1 to 5 mg per kg of body weight. In order tointroduce the plasmids into cells in vitro, the content of the vectorsfor use is 1 to 5 μg/10⁶ cells. Herein below, the present invention willbe specifically described with reference to Examples.

All prior art documents cited herein are incorporated by reference intheir entirety.

EXAMPLES Example 1 A. Analysis of Expression of ILT7 A-1) Analysis UsingSAGE Library

The expression of genes in human monocytes, IPCs, and HSV-treated IPCswas compared and analyzed by Serial Analysis of Gene Expression (Tradename; SAGE) method. The analysis method is as follows. Monocytes wereseparated as BDCA-4 positive cells and IPCs were separated as CD14positive cells from human peripheral blood using a cell sorter. Further,IPCs were cultured for 12 hours under the presence of Herpes SimplexVirus (HSV) and then the differentiated IPCs were prepared. RNAs wereobtained from respective cells, followed by producing a SAGE libraryusing I-SAGE (Trade name) kit (manufactured by Invitrogen). Data on theobtained base sequences of about 100,000 tags was analyzed using SAGEAnalysis Software (manufactured by Invitrogen). As a result, a genewhose score value of monocyte/IPC/IPC+HSV is 0/16/0, namely, ILT7 (GenBank Acc#NM_012276) known as a gene, which shows IPC specificexpression, was found. ILT7 is a membrane protein withimmunoglobulin-like domains encoded by a base sequence shown in SEQ IDNO: 1 (FIG. 2 (a)). It has been reported that mRNA of ILT7 is expressedin IPCs (Blood 100, 3295-3303 (2002)).

A-2) RT-PCR

The expression of ILT7 in hemocyte cells was examined in more detail.Each cell was preparatively isolated from human peripheral blood by thecell sorter. RNAs were extracted from each of the isolated cellpopulations, from which cDNA was synthesized. Quantitative PCR wasperformed in accordance with an ordinary method using the resulting cDNAas a template and the expression level of mRNA of ILT7 was analyzed. Theused conditions for the base sequences of primers and PCR are asfollows:

Forward primer: 5′ CTC CAA CCC CTA CCT GCT GTC 3′ (SEQ ID NO: 3)Reverse primer: 5′ TTC CCA AGG CTC CAC CAC TCT 3′ (SEQ ID NO: 4)1 cycle of PCR (at 94° C. for 3 minutes)25 cycles of PCR [at 94° C. for 30 seconds, at 58° C. for 30 seconds,and at 72° C. for 1 minute]1 cycle of PCR (at 72° C. for 6 minutes)

When IPCs stimulated by monocytes, IPCs, HSVs, and CD19 positive cells(i.e. B cells), CD3 positive cells (i.e. T cells), T cells stimulated byPMAs, and CD56 positive cells (i.e. NK cells) were examined, it wasfound that ILT7 was expressed specifically in IPCs (FIG. 1 (a)).

A-3) Quantitative RT-PCR

Further, the expression in other organs and tissues was examined byquantitative PCR using ABI PRISM 7000 (manufactured by AppliedBiosystem). As cDNA panel, BD (Trade name) MTC multiple tissue cDNApanel (Human I; Cat. No. 636742, Human immune; Cat. No. 636748, Humanblood fractions; Cat. No. 636750; all of them are manufactured by BectonDickinson) and the same cDNA derived from hemocyte cells as described in2) were used.

The used base sequences of primers are as follows:

Forward primer for ILT7:  (SEQ ID NO: 5) 5′CCT CAA TCC AGC ACA AAA GAA GT 3′ Reverse primer for TLT7: (SEQ ID NO: 6) 5′ CGG ATG AGA TTC TCC ACT GTG TAA 3′Forward primer for GAPDH:  (SEQ ID NO: 7) 5′ CCA CCC ATG GCA AAT TCC 3′Reverse primer for GAPDH:  (SEQ ID NO: 8) 5′TGG GAT TTC CAT TGA TGA CAA G 3′

PCR was performed by using ABI PRISM 7000 (manufactured by AppliedBiosystem) and SYBR green PCR master mix kit (manufactured by the samecompany). Sequence Detection System Software (manufactured by the samecompany) was used for analysis.

The reaction conditions are as follows:

Step 1: 1 cycle of PCR (at 50° C. for 2 minutes)Step 2: 1 cycle of PCR (at 95° C. for 10 minutes)Step 3: 40 cycles of PCR (at 95° C. for 15 seconds, at 60° C. for 1minute)

The expression of ILT7 gene was compared between each tissue bystandardizing at the level of expression of theglyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, which is known tobe expressed constitutively. As a result, it was observed that ILT7 wasnot expressed in any organs other than lymphoid tissues and expressedspecifically in IPCs.

B. Production of ILT7 and FcRγ Expression Vectors

Subsequently, cloning of genes and production of expression vectors werecarried out in order to express ILT7 proteins.

B-1) Cloning of ILT7 Genes

Poly(A) ⁺RNA separated from human peripheral blood was extracted fromIPCs, from which cDNA was synthesized using oligo dT primer and SuperScript Choice System for cDNA Synthesis kit. An EcoRI adapter wasligated into the synthesized cDNA, which was ligated into pME18S vectorcleaved by EcoRI, resulting in production of human IPC cDNA library.ILT7 gene was amplified by PCR method using the produced cDNA library asa template as well as using primers with the following base sequences. 1unit of KOD Plus DNA polymerase (manufactured by TOYOBO CO., LTD.) wasused for PCR reaction. Reaction conditions were set to 25 cycles of PCR[at 94° C. for 15 seconds, at 55° C. for 30 seconds, and at 68° C. for 2minutes] after 1 cycle of PCR at 94° C. for 2 minutes.

Forward primer: 5′ CAG GGC CAG GAG GAG GAG ATG 3′ (SEQ ID NO: 9)Reverse primer: 5′ TCA GCA GAC ACT TCC CCA ACT 3′ (SEQ ID NO: 10)

The 2-kb ILT7 cDNA fragment amplified was separated and recovered byelectrophoresis using 1% agarose gel, which was cloned to pCR4Blunt-TOPO plasmid vector (manufactured by Invitrogen) using Zero BluntTOPO PCR Cloning kit (manufactured by Invitrogen). The base sequences ofthe genes obtained was analyzed, and it was found that the desired ILT7gene shown in SEQ ID NO: 1 was obtained.

B-2) Production of FLAG-Tagged ILT7 Expression Vectors

A plasmid expressing a protein in which FLAG tags were fused to N- andC-termini of ILT7, respectively was constructed. ILT7 was fused with atag, which allowed for confirming the expression of ILT7 protein bydetection of the tag. The desired sequence was amplified by PCR methodusing the ILT7 gene produced as described in 1) as a template as well asusing primers with the following base sequences. 1 unit of KOD Plus DNApolymerase (manufactured by TOYOBO CO., LTD.) was used for PCR reaction.Reaction conditions were set to 25 cycles of PCR [at 94° C. for 15seconds, at 55° C. for 30 seconds, and at 68° C. for 2 minutes] after 1cycle of PCR at 94° C. for 2 minutes.

For N-FLAG ILT7 Forward primer (SEQ ID NO: 11):  5′CCG ctc gag ATG ACC CTC ATT CTC ACA AGC CTG CTCTTC TTT GGG CTG AGC CTG GGC [GAT TAC AAG GAT GAC GAC GAT AAG]CCC AGG ACC CGG GTG CAG GCA GAA 3′ Reverse primer (SEQ ID NO: 12):  5′C TAG act agt TCA GAT CTG TTC CCA AGG CTC 3′ For C-FLAGILT7Forward primer (SEQ ID NO: 13):  5′CCG ctc gag ATG ACC CTC ATT CTC ACA AGC 3′Reverse primer (SEQ ID NO: 14): 5′C TAG act agt TCA [CTT ATC GTC GTC ATC CTT GTA ATC]GAT CTG TTC CCA AGG CTC 3′

In the above-mentioned base sequences, each underlined portion inparentheses shows a base sequence encoding the attached FLAG tag andeach lowercase letter shows the cleavage site for the restriction enzymeXhoI or SpeI. DNA fragments amplified by PCR were cleaved by XhoI andSpeI, which were then separated by Gel electrophoresis. 2 kb DNAfragments were recovered, which were ligated into pME18X vector cleavedby XhoI and SpeI in the same manner as described above. Then, two typesof plasmids capable of expressing the desired fusion protein, i.e.pME18X-N-FLAG ILT7 and pME18 X-C-FLAG ILT7 were constructed,respectively.

B-3) Cloning of FcRγ Genes

FcRγ protein was considered as a protein capable of associating withILT7 protein. The present molecule is a gene with base sequences andamino acid sequences of SEQ ID NO: s 15 and 16 (Genbank Acc#NM_004106,J. Biol. Chem. 265, 6448-6452 (1990)). The molecule is a molecule (γchain) which constitutes FcεRI, i.e. a high affinity IgE receptor.Although it is also named as FcεRIγ, it will be referred to as FcRγhereinafter. In this regard, the present molecule has also been known asa component of FcγR or FcαR. The present gene was cloned by PCR methodas shown below to produce expression vectors. FcRγ gene was amplified byPCR method using the human IPC cDNA library produced as described in 1)as a template as well as using primers with the following basesequences. 1 unit of KOD Plus DNA polymerase (manufactured by TOYOBOCO., LTD.) was used for PCR reaction. Reaction conditions were set to 25cycles of PCR [at 94° C. for 15 seconds, at 55° C. for 30 seconds, andat 68° C. for 1 minute] after 1 cycle of PCR at 94° C. for 2 minutes.

Forward primer: (SEQ ID NO: 17) 5′ CCC AAG ATG ATT CCA GCA GTG 3′Reverse primer: (SEQ ID NO: 18) 5′ GGA AGA ACC AGA AGC CAA AGA 3′

The 0.3-kb FcRγ cDNA fragment amplified was separated and PGP-27 DNArecovered by electrophoresis using 2% agarose gel, which was cloned topCR4 Blunt-TOPO plasmid vector (manufactured by Invitrogen) using ZeroBlunt TOPO PCR Cloning kit (manufactured by Invitrogen). The basesequences of the genes obtained were analyzed, and it was confirmed thatthe desired FcRγ gene shown in SEQ ID NO: 15 was cloned.

B-4) Production of Myc-Tagged FcRγ Expression Vectors

A plasmid expressing a protein in which Myc tag was attached to Cterminus was constructed so that the expression of FcRγ protein could beconfirmed. The desired sequence was amplified by PCR method using theFcRγ gene produced as described in 3) as a template as well as usingprimers with the following base sequences. 1 unit of KOD Plus DNApolymerase (manufactured by TOYOBO CO., LTD.) was used for PCR reaction.The conditions were set to 25 cycles of PCR [at 94° C. for 15 seconds,at 55° C. for 30 seconds, and at 68° C. for 1 minute] after 1 cycle ofPCR at 94° C. for 2 minutes.

Forward primer (SEQ ID NO: 19):  5′CCG ctc gag ATG ATT CCA GCA GTG GTC TTG 3′Reverse primer (SEQ ID NO: 20):  5′CTA Gac tag tCT A[CA GAT CCT CTT CAG AGA TGAGTT TCT GCT C]CT GTG GTG GTT TCT CAT G 3′

Of the above-mentioned primer sequences, the underlined portion inparentheses shows a base sequence encoding the attached Myc tag and eachlowercase letter shows the cleavage site for the restriction enzyme XhoIor SpeI. DNA fragments amplified by PCR were cleaved by XhoI and SpeI,which were then separated by Gel electrophoresis. About 0.3-kb DNAfragments were recovered, which were ligated into pME18X vector cleavedby XhoI and SpeI in the same manner as described above. Then, a plasmidcapable of expressing the desired fusion protein, i.e. pME18X-Myc-FcRγwas constructed.

C. Expression of ILT7 in Animal Cells

The expression of ILT7 in animal cells was examined using expressionvectors produced as described above.

C-1) Expression in 293T Cells

DNAs consisting of the following five combinations were introduced into293T cells (7×10⁵ cells) using effectene transfection kit (manufacturedby Qiagen). Two days after the introduction, flow cytometry analysis(FCM analysis) was carried out.

(1) pME18X-N-FLAG ILT7 2 μg(2) pME18X-C-FLAG ILT7 2 μg(3) pME18X-N-FLAG ILT7 1 μg+pME18X-Myc-FcRγ 1 μg(4) pME18X-C-FLAG ILT7 1 μg+pME18X-Myc-FcRγ 1 μg(5) pME18X-Myc-FcRγ 2 μg

The method of FCM analysis was performed in the same manner as describedin A-4 of the following Example 2. Cy3 conjugated anti-Flag antibody(manufactured by Sigma) was used for the reaction and FACScan(manufactured by Becton Dickinson) was used for the analysis. As aresult, it was found that only a few ILT7 was expressed on the cellsurface when reacted alone, while ILT7 was expressed extracellularly androbustly when coexisted with FcRγ (FIG. 3). It is known that mouse FcRγhas high homology with human FcRγ. However, when p815 cells (mousemastocytoma) which express mouse FcRγ were used as hosts, the expressionof ILT7 could not be observed.

C-2) Analysis by Immunoprecipitation and Western Blotting Method

ILT7 was expressed with accompanying FcRγ on the cell surface, which wasconfirmed as follows. After immunoprecipitation, various antibodies foreach 293T cell which was coexpressed with both genes in respectivecombinations described in (1) to (5) were analyzed. DNAs were introducedinto 293T cells (7×10⁵ cells), from which 293T cells were recovered twodays after the introduction in the same manner as described in 1). Cellfractionations were dissolved in lysis buffer (0.5% Triton, 150 mMNacl), which was left on ice for 20 minutes. Thereafter, aspirationusing a needle (27G) was repeated several times, followed bycentrifuging at 15 Krpm for 20 minutes. Anti-myc antibody (2 μg,manufactured by Santa cruz biotechnology) or anti-Flag antibody (2 μg,manufactured by Sigma) was added to 200 μg of lysate of the resultingproducts, which was further stirred by rotation at 4° C. for 4 hours.Then, Protein A/G Sepharose 4 Fast Flow mix (manufactured by Amershambioscience) was added thereto, which was stirred by rotation at 4° C.for 1 hour. Then, the resulting precipitated fractions were washed withlysis buffer with the following composition 3 times.

Lysis Buffer: 0.5% Triton X-100, 50 mM HEPES (pH 7.6), 150 mM NaCl, 1 mMEDTA,

10% glycerol,

1 mM DTT, 2 mM PMSF,

1 μg/ml Aprotinin,1 μg/ml Leupeptin,1 μg/ml Pepstatin A,0.1 μg/ml Chymostatin,1 mM Na₃VO₄,0.1 mM β-glycerophosphate

A sample buffer for SDS-PAGE was added to the washed precipitates, whichwas boiled for 5 minutes and centrifuged, followed by performingelectrophoresis with 10% SDS gel. Samples were transferred from gelsafter electrophoresis to PVDF membrane (Immobilon-p-transfer membrane:manufactured by Millipore) in accordance with an ordinary method.Blotting was performed with anti-Flag antibody and anti-myc antibody. Itwas confirmed that the ILT7 associated with FcRγ was present in 293Tcells because their presence in each immune precipitate was observed(FIG. 4).

C-3) Analysis of Sugar Chain

Since several bands of ILT7 were observed in the Western analysis, thepossibility that ILT7 was glycosylated was examined. 200 μg of lysate of293T cells which express N-FLAG ILT7 and Myc-FcRγ was immunoprecipitatedwith anti-Flag antibody in the manner as described in 1) and 2).Thereafter, the precipitated fractions were suspended in 60 μL ofN-glycosidase buffer with the following composition and 30 μL of eachresulting solution was aliquoted into two tubes.

N-Glycosidase Buffer: 10 mM EDTA, 0.2% SDS, 0.5% Triton X100,

1% 2-mercaptoethanol in PBS (phosphate buffer)

Then, 3 units of 3 μL of N-glycosidase (#1365177, manufactured by Roche)were added to one tube, which was reacted at 37° C. for 15 hours.Further, 7 μL of sample buffer was added thereto, which was heated at100° C. for 5 minutes, followed by performing electrophoresis with 10%SDS gel. After electrophoresis, gel was transferred to PVDF membrane, towhich 1 μg of anti-ILT7 polyclonal-antibody as described in 4) was addedand reacted at 4° C. overnight. The resulting product was washed withTBS-T buffer and reacted with 100,000-fold diluted HRP-labeledanti-rabbit antibody (manufactured by Jackson) at room temperature.Then, it was colored with ECL Western Blotting Detection System(manufactured by Amersham bioscience). As a result, the apparentmolecular weight was decreased by performing N-glycosidase treatment.Thus, it was expected that sugar chains were added to ILT7 (FIG. 5).

C-4) Production of Anti-ILT7 Polyclonal Antibody

The used anti-ILT7 polyclonal antibody as described in 3) was producedas follows. Peptide of 23 amino acids corresponding to C terminus ofILT7 (CSQEANSRKDNAPFRVVEPWEQI; SEQ ID NO: 21) was chemically synthesizedand bound to KLH protein which is a career, and the resulting productwas used as an immunogen. Rabbits were intradermally immunized withimmunogen mixed with Freund complete adjuvant. After six immunizationsin all (once per week), the increased antibody titer in serum wasconfirmed and then whole blood was collected. Then, some serum wasaffinity purified using peptide column of the same sequence. Theresulting product was determined as anti-ILT7 polyclonal antibody.

Example 2 A. Production of Anti-ILT7 Monoclonal Antibody A-1) Productionof Immunogen

Cells to be used as immunogens were prepared by introducing genes to293T cells as described below. 46.4 μg of transgene (pME18 X-C-FLAG ILT723.2 μg and pME18X-Myc-FcRγ 23.2 μg) was added to the bottom of 100mm/Collagen Coated Dish (IWAKI) coated with 3 mL of opti-MEM (GIBCO) andmixed. Subsequently, aside from the transgene solution, 58 μL ofLipofectamine (Trade name) 2000 (Invitrogen) was diluted with 3 mL ofopti-MEM, which was allowed to stand at room temperature for 5 minutesand Lipofectamine solution was prepared. Thereafter, Lipofectaminesolution was gently added to the dish containing the transgene solutionand mixed. After standing at room temperature for 20 minutes, 10 mL of293T-cells, diluted to 1×10⁶ cells/ML using DMEM culture medium (SIGMA)containing 10% FBS (fetal bovine serum), was gently added to the dish.The resulting medium was subjected to static culture in an incubator at37° C. under CO₂ for 48 hours, from which cells were recovered bypipetting. The obtained cells were used as transfectants for immunogens.

A-2) Production of hybridomas

On the day before cells were immunized, 50 μL of emulsion obtained bymixing 200 μL of PBS with 200 μL of complete adjuvant (FREUND) (RM606-1,manufactured by Mitsubishi Kagaku Iatron, Inc.) was injected to thebottoms of both feet of four Balb/c female mice (four-week-old) forimmunization. On the following day, 50 μL of 2×10⁷ cells suspended in400 μL of PBS was immunized. The second and third immunizations wereperformed every four days. Three days following the third immunization,cell fusion was performed as follows. Cells were collected from lymphnodes of mice feet immunized. Mouse myeloma cells P3-X63-Ag8-U1 culturedin RPMI1640 culture medium (SIGMA) containing 10% FBS were mixed withthe cells derived from lymph nodes and myeloma so that the ratio of themouse myeloma cells to the cells derived from lymph nodes and myelomashould be 2:1 to 10:1, from which cells were recovered bycentrifugation. PEG4000 (MERCK) equivalently diluted with RPMI1640culture medium was added to the obtained cell fractions, which wassubjected to cell fusion. After washing cells, the resulting product wassuspended in 160 mL of 15% FBS-HAT-medium containing a supplement andthen inoculated into sixteen 96-well plates at 200 μL/well. The culturemedium was exchanged after three days. One to two weeks afterobservation of the colony formation, primary screening was performed.

A-3) Screening of Hybridoma by Cell ELISA Method

Hybridoma, which produces target antibody, was screened by the followingCell ELISA. The produced cells as described in 1) were used at 1×10⁷cells per 96-well plate, which were suspended in 0.5% BSA/2 mM EDTA/PBSand then aliquoted to a plate for Cell ELISA (NUNC 249570 96V NW PS) at100 μL/well. Centrifugation was carried out at 2,000 rpm at 4° C. andthen the supernatant was discarded. Sampled culture supernatant wasadded at 50 μL/well, which was reacted at room temperature for 30minutes. Washing operation that involves adding 0.5% BSA/2 mM andEDTA/PBS to each well, centrifuging at 2,000 rpm at 4° C. for 2 minutes,and then discarding the supernatant was carried out twice. 50 μL/well of10,000-fold diluted peroxidase-labeled goat anti-mouse IgG antibody(IM0819; Beckman coulter) was added to each well after washing, whichwas reacted for 30 minutes. The washing operation using 0.5% BSA/2mM-EDTA/PBS was carried out twice, followed by adding a coloringsolution. The prepared antibody solution was substituted with PBS (−) bya dialysis membrane (10,000 cuts manufactured by PIERCE) to givepurified anti-ILT7 chimeric antibodies.

A-4) Examination of Antibody Responsiveness by Flow Cytometry (FCM)Analysis

Hybridoma culture supernatant was analyzed by flow cytometry (FCM)analysis. The produced cells as described in 1) was suspended in 0.5%BSA/2 mM EDTA/PBS, which was transferred into a centrifugal tube at1×10⁵ per one sample, followed by adding 40 μL of each culture andreacting at room temperature for 30 minutes. Washing operation thatinvolves adding 1 ml of 0.5% BSA/2 mM and EDTA/PBS to each tube,centrifuging at 1200 rpm at 4° C. for 3 minutes, and discarding thesupernatant was carried out twice. 40 μL of 100-fold dilutedFITC-labeled goat anti-mouse IgG antibody (IM0819; Beckman coulter) wasadded to each well after washing, which was reacted at room temperaturefor 30 minutes. The washing operation using 0.5% BSA/2 mM-EDTA/PBS wascarried out twice, followed by analyzing using flow cytometry FC500(Beckman coulter). A hybridoma producing an antibody which did notrespond to only host cell and responded specifically to the cell intowhich gene had been introduced was selected. The selected hybridoma wascloned by the limiting dilution method and hybridomas #11 and #17 whichproduce monoclonal antibodies were obtained.

B. Examination of Responsiveness of Anti-ILT7 Antibody

ILT7 in which FLAG tag was attached to N terminus was coexpressed withFcRγ molecule in 293T cells in the same manner as described in C-1) ofExample 1. Then, the responsiveness of the antibody obtained in Example2 was confirmed by FCM analysis using FACScan (Becton Dickinson). As aresult, it was confirmed that all antibodies produced by hybridomas #11and #17 which were obtained as described in A responded to the cellsinto which ILT7 gene was introduced and which expressed ILT7 (FIG. 6(b)). Further, lymphocytes were separated from human peripheral bloodusing Ficoll and then double staining with the produced anti-ILT7antibody and PE-labeled anti-BDCA-2 antibody (Miltenyi) was performed.Then, the responsiveness to the lymphocytes was examined. As a result,the binding of monoclonal antibody produced by hybridomas #11 and #17 toBDCA-2 positive cell was detected. That is, it was confirmed that bothmonoclonal antibodies recognized ILT7 molecules expressed on human IPCs(FIG. 6 (a)). These monoclonal antibodies were designated as anti-ILT7antibody #11 and anti-ILT7 antibody #17, respectively. More detailedanalysis was performed.

Multiple-staining analysis for human peripheral blood lymphocytes wascarried out using the produced anti-ILT7 antibody, anti-Lineage-1antibody (anti-CD3, CD14, CD16, CD19, CD56 antibodies; BectonDickinson), anti-CD123 antibody (Becton Dickinson), and anti-BDCA-2antibody (Miltenyi). As for ILT7 antibody-positive fractions, LineageMarker was negative, CD123 was positive, and BDCA-2 was positive. Fromthe results, it was confirmed that IPCs were stained by only ILT7#11 andILT7#17 (FIG. 7).

Further, the expression of various molecules was examined by FCManalysis when human peripheral blood lymphocytes were stimulated by CpGor IFNα for 24 hours. CpGODN2216 was used as CpGA, which induces theproduction of IFN from IPCs and CpGODN2006, was used as CpGB whichfacilitates the maturation of dendritic cells (Moseman et al. J.Immunology. 173, 4433-4442, 2004). A gate was set to Lineage Markernegative fraction. When the responsiveness of anti-BDCA-2 antibody andanti-ILT7 antibody to CD123 positive cell population was analyzed, mostof the ILT7 positive fractions were disappeared even after 24-hours CpGstimulation. On the other hand, some cells of BDCA-2 showed positiveafter 24-hours CpG stimulation (FIG. 8). It has been considered thatIPCs are differentiated into different cells immediately after the CpGstimulation. It was indicated that the anti-ILT7 antibody of the presentinvention was useful as a stage specific antibody to IPCs. Further, itwas confirmed that IPCs in peripheral blood lymphocytes were notdifferentiated under the presence of IFNα, in this case where thesurvival ratio was high, the expression of ILT7 was maintained on IPCs,further ILT7 was stably present on IPCs in autoimmune diseases with thepossibility that IFN in serum was at a high level.

C. Examination of Specificity of Anti-ILT7 Antibody

ILT7 belongs to ILT/LIR family and there is a plurality of moleculeswith high homology, particularly with high homology in the extracellularregion (FIG. 9). It has been reported that mRNAs of molecules,especially such as ILT2 and ILT3 are expressed in IPCs (Ju et al. Gene331, 159-164, 2004). Therefore, the responsiveness of these moleculeswas confirmed using transgenic cells.

C-1) Cloning of ILT1 Molecule and Production of Expression Vectors

cDNA was synthesized from RNA derived from human tonsil using oligo dTprimer and SuperScript Choice System for cDNA Synthesis kit. Next, aNotI adapter was ligated into pME18S vector cleaved by NotI, resultingin production of human tonsil cDNA library.

ILT1 gene with a FLAG tag at the C terminus was amplified by PCR methodusing the produced cDNA library as a template as well as using primerswith the following base sequences. 1 unit of KOD Plus DNA polymerase(manufactured by TOYOBO CO., LTD.) was used for PCR reaction. Reactionconditions were set to 25 cycles of PCR [at 94° C. for 15 seconds, at55° C. for 30 seconds, and at 68° C. for 2 minutes] after 1 cycle of PCRat 94° C. for 2 minutes.

Forward primer (SEQ ID NO: 22):  5′CCG ctc gag ATG ACC CCC ATC CTC ACG GTC C 3′Reverse primer (SEQ ID NO: 23):  5′CTA Gac tag tTC A[CT TAT CGT CGT CAT CCT TGTAAT C]CC TCC CGG CTG CAT CTT G 3′

In the above-mentioned primer sequences, the underlined portion inparentheses shows a base sequence encoding the attached FLAG tag andeach lowercase letter shows the cleavage site for the restriction enzymeXhoI or SpeI. DNA fragments amplified by PCR were cleaved by XhoI andSpeI, which were then separated by Gel electrophoresis. About 2-kb DNAfragments were recovered, which were ligated into pME18X vector cleavedby XhoI and SpeI in the same manner as described above. Then, a plasmidcapable of expressing the desired fusion protein, i.e. pME18X-C-FLAGILT1was constructed. The base sequence and amino acid sequence are shown inSEQ ID NO: s: 24 and 25.

C-2) Production of Expressing Cells and Examination of AntibodyResponsiveness

As for ILT2 (SEQ ID NO: 26) and ILT3 (SEQ ID NO: 28), expression vectorsin which respective genes were cloned to XbaI or XhoI sites of pcDNA4.1(manufactured by Invitrogen) were used. DNAs of the followingcombinations were introduced into 293T cells (7×10⁵ cells) in the samemanner as described in C-1). Two days after the introduction, FCManalysis was carried out and then anti-ILT7 antibody was analyzed.

(1) pME18X-N-FLAG ILT7 1 μg+pME18X-Myc-FcRγ 1 μg(2) pME18X-C-FLAG ILT1 0.5 μg+pME18X-Myc-FcRγ 0.5 μg+pcDNA4.1-ILT2 0.5μg+pcDNA4.1-ILT3 0.5 μg

As a result, any antibodies did not respond to the cells in which ILT1was expressed. For this reason, it was suggested that these anti-ILT7antibodies specifically recognized ILT7 molecules on IPCs (FIG. 10).

Example 3 Effect of Anti-ILT7 Antibody on Ability to Produce Human IFN

Human peripheral blood lymphocytes were inoculated into 96 well plate at2×10⁵ cells/well, which were reacted with 5 μg/mL of various antibodiesat 37° C. After 1-hour culture, influenza virus PR8 was added thereto.After 24-hours culture, IFNα in the culture supernatant was measured byELISA kit (Bender Med System). As a result, the production of IFN wasinhibited by the addition of anti-ILT7 antibody (FIG. 11). Namely, itwas found out that the IFN production by IPCs was affected by theanti-ILT7 antibody of the present invention.

Example 4 CDC Activity of Anti-ILT7 Antibody

A. Production of anti-ILT7 monoclonal antibody

A clone which produces a monoclonal antibody was obtained in the samemanner as described in A-1) to A-4) of Example 2. The responsiveness wasexamined in the same manner as described in B of Example 2 and thespecificity was examined in the same manner as described in C of Example2. As a result, hybridomas #37, #28, and #33, which produced anti-ILT7monoclonal antibodies with good responsiveness and specificity, wereobtained. CDC activity was measured as described below using anti-ILT7monoclonal antibody in which three kinds of these hybridomas wereproduced.

B. Determination of CDC Activity

B-1) On the previous day of production of target Production of targetcell line (ILT7-CHO cell line), the following DNA was introduced intoCHO-k1 cells, which were inoculated so as to be 6×10⁵ cells per one dish(6 cmφ) using Effectene Transfection Reagent (manufactured by QLAGEN)and then resistant strains were selected using 800 μg/ml of Zeocin(manufactured by Invitrogen).Introduced DNA: pcDNA3.1-C-FLAG ILT7 1 μg+pME18X-Myc FcRγ 2 μg

Thereafter, a cell line, which highly expressed ILT7, was obtained usingthe cell sorter (BD FACSAria, manufactured by Becton Dickinson). It wasconfirmed that the selected cell line highly expressed ILT7 by FCManalysis. Operation of FCM analysis was carried out in accordance withthe method as described in A-4) of Example 2 except that BD FACSCaliber(manufactured by BD) was used for FCM. The following antibodies wereused for a primary antibody and a secondary antibody, respectively.

Primary antibody: 5 μg/ml mouse anti-ILT7 antibody (#37),

Secondary antibody: R-phycoerythrin (R-PE)-conjugated goat anti-mouseimmunoglobulin specific polyclonal antibody (BD)

B-2) Response of Target Cells to Anti-ILT7 Antibodies

The obtained target cells as described in B-1) (ILT7-CHO cell) wererecovered using 5 mM EDTA/PBS solution, which were suspended in CDCmedium with the following composition so as to be a concentration of4×10⁵ cells/ml. The suspension was aliquoted into each 96-well plate at50 μl/well.

CDC Medium: RPMI11640 0.1% BSA

100 units/ml Penicillin100 μg/ml Streptomycin

10 mM Hepes (pH 7.6) 2 mM L-Glutamin

50 μl of anti-ILT7 antibody solution prepared by CDC medium was added toeach well and mixed so that the final concentration of antibodies shouldbe 0.1 μg/ml, 0.5 μg/ml, 1 μg/ml, and 5 μg/ml. Further, 50 μl of CDCmedium containing a complement with the following composition was addedthereto and mixed so that the final complement concentration should be6%, followed by culturing at 37° C. for 2 hours.

CDC Medium Containing a Complement:

1 ml of complement of juvenile rabbit (Catalog No.: CL3441, manufacturedby CEDARLANE)CDC medium (vide supra)

Then, the suspension was centrifuged (centrifugal condition: at 250 Gfor 4 minutes) and the supernatant was recovered while paying attentionnot to be contaminated with cells. LDH in the supernatant was measuredby an ordinary method, which was determined as “The amount of LDH leakedfrom the target cell by the complement activity” (Experimental Sample).

The following parameters were also prepared in order to determine CDCactivity.

Target Cell Spontaneous LDH Release: only target cells were cultured inthe same volume as the sample and prepared.

Target Cell Maximum LDH Release: only target cells were cultured in thesame volume as the sample, and then Triton X-100 solution included withthe kit was added thereto 60 minutes before recovery of the supernatantso that the final concentration should be 0.8% and prepared.

Volume Correction Control: the same amount of Triton X-100 as that addedwhen Target Cell Maximum LDH Release was prepared was added to theculture medium of the same volume as the sample and prepared.

Culture Medium Background: the culture medium of the same volume as thesample and the solution to which complement containing CDC medium wasadded to the culture medium so as to be the same volume as the samplewere prepared.

The same volume of culture medium as the sample was subtracted from theabsorbance of Target Maximum and Target Spontaneous. The solution towhich complement containing CDC medium was added to the culture mediumso as to be the same volume as the sample was subtracted from theabsorbance of Experimental Sample and corrected. The CDC activity wascalculated by the following equation. The results are shown in Table 1and FIG. 12. Even in the case where anti-ILT7 monoclonal antibodiesobtained from any hybridoma were used, 80% or more of CDC activity wasexhibited when the antibody concentration was 0.5 μg/ml or more.

${{CDC}\mspace{14mu} {activity}\mspace{14mu} (\%)} = {\frac{\begin{matrix}{{{Experimental}\mspace{14mu} {Sample}} -} \\{{Target}\mspace{14mu} {Spontaneous}}\end{matrix}}{\begin{matrix}{{{Target}\mspace{14mu} {Maximum}} - {{Volume}\mspace{14mu} {Control}} -} \\{{Target}\mspace{14mu} {Spontaneous}}\end{matrix}} \times 100}$

TABLE 1 Antibody concentration Cytotoxicity Cytotoxicity (μg/ml) (Aver)(STD) #37 0.1 14.78 3.16 0.5 85.50 0.60 1 86.13 2.93 5 90.26 1.87 #280.1 18.52 0.60 0.5 80.97 1.62 1 83.64 1.99 5 88.17 3.32 #33 0.1 4.421.58 0.5 82.16 3.35 1 85.39 2.78 5 86.18 1.71 Mouse 0.1 1.53 0.60 IgG2a0.5 1.47 2.50 1 3.68 2.90 5 3.06 1.72 no Ab 0 2.10 0.49

Comparative Example 1

Exactly the same operation was performed in the same manner as describedin B and C of Example 4 except that mouse IgG2a was used in place ofanti-ILT7 antibody. The results are shown in Example 4 as well as Table5 and Fig. The CDC activity to the target cells was not observed inantibodies other than anti-ILT7 monoclonal antibody.

Example 5 Internalization of Anti-ILT7 Antibody to Target Cells A.Anti-ILT7 Monoclonal Antibody

The following anti-ILT7 monoclonal antibodies were used. Anti-ILT7monoclonal antibodies: #17, #26, #37, #28, and #33

B. Observation of Internalization B-1) Production of Target Cell Line(ILT7-CHO Cell Line)

The target cell line (ILT7-CHO cell line) was produced in the samemanner as described in B-1 of Example 4.

B-2) Response of Target Cells to Anti-ILT7 Antibody

The recovered ILT7-CHO cells were suspended in ice-cold buffer (T(−)+10%FBS) with the following composition at 1×10⁶ Cells/mL using 5 mM ofEDTA/PBS solution.

T (−) Medium: RPMI1640

100 units/ml Penicillin100 μg/ml Streptomycin

10 mM Hepes (pH 7.6) 2 mM L-Glutamin

1 mM sodium pyruvate50 μM 2-mercaptoethanol10% heat inactivated Fetal Bobine Serum1 mL of suspension as described above was placed into a 15 mLcentrifugal tube, which was centrifuged (centrifugal condition: at 1200rpm, at 4° C., for 5 minutes) and then the supernatant was discarded.200 μL of anti-ILT7 monoclonal antibody suspension of (10 μg/mL) wasadded to cell pellets, which was mixed and incubated at 4° C. for 30minutes, followed by washing with ice-cold T (−) medium twice (theamount of the medium used: 10 mL per washing, centrifugal condition: at1200 rpm, at 4° C., for 5 minutes).

B-3) Modification of ILT7-Anti-ILT7 Antibody Immune Complex Present onthe Surface of Target Cells

Subsequently, ILT7-anti-ILT7 antibody immune complex present on thesurface of cells was modified with a secondary antibody, which waslabeled with fluorescence for detection. Specific method is describedbelow. APC-labeled goat anti-mouse IgG polyclonal antibody (Catalognumber: 550826BD, manufactured by Biosciences) containing ice-cold T (−)medium was added to cell pellets obtained as described in B-2), whichwas incubated with shading at 4° C. for 20 minutes, followed by washingwith ice-cold T (−) medium twice (the amount of the medium used: 10 mLper washing, centrifugal condition: at 1200 rpm, at 4° C., for 5minutes). Then, ice-cold T (−) medium was added thereto, which was usedas 1×10⁶ Cells/mL of suspension.

B-4) Induction of Internalization by Incubation at 37° C.

The suspension obtained as described in B-3 was equally divided into twotubes (i.e. tubes (a) and (b)). The tubes (a) and (b) were incubated at37 and 4° C., respectively, under shading condition for 60 minutes.After the incubation, 1% FBS/PBS (ice-cold) was added thereto in orderto stop internalization. The resulting solution was centrifuged(centrifugal condition: at 1200 rpm, at 4° C., for 5 minutes) and thenthe supernatant was discarded, followed by washing with 1% of FBS/PBS(ice-cold) twice (the amount of the solution: 10 mL per washing,centrifugal condition: at 1200 rpm, at 4° C., for 5 minutes).

B-6) Modification of ILT7-Anti-ILT7 Antibody Immune Complex Remained onthe Surface of Target Cells after Incubation

ILT7-anti-ILT7 antibody immune complex remained on the cell surfaceafter incubation was modified with a tertiary antibody in order todetect, by fluorescence. Specific method is described below. 20 μL ofsuspension containing tertiary antibodies (FITC-labeled donkey anti-goatIgG antibody (Catalog number: sc-2024, manufactured by Santa cruzbiotechnology)) was added to cell pellets obtained as described in B-4),which was mixed and allowed to stand at 4° C. for 15 minutes undershading condition. The resulting solution was washed with (the amount ofthe solution: 10 mL per washing, centrifugal condition: at 1200 rpm, at4° C., for 5 minutes).

B-5) Analysis of Anti-ILT7 Antibody Present in Target Cells

Subsequently, 150 μL of 1% FBS/PBS was added to cell pellets obtained asdescribed in B-5), which was suspended and collected into a 1.2 mlmicrotiter tube, followed by performing FCM analysis. In analysis, themean fluorescence intensity (MPI) of each cell was analyzed separatelyin FITC and APC. Further, the fluorescence intensity ratio (%) wascalculated by the following equation.

${\begin{matrix}{Fluorescence} \\{{intensity}\mspace{14mu} {ratio}}\end{matrix}(\%)} = {\frac{\begin{matrix}{{Mean}\mspace{14mu} {fluorescence}\mspace{14mu} {intensity}\mspace{14mu} {of}\mspace{14mu} {cells}} \\{{incubated}\mspace{14mu} {at}\mspace{14mu} 37{^\circ}\mspace{14mu} {C.\mspace{14mu} {for}}\mspace{14mu} 60\mspace{14mu} {minutes}}\end{matrix}}{\begin{matrix}{{Mean}\mspace{14mu} {fluorescence}\mspace{14mu} {intensity}\mspace{14mu} {of}\mspace{14mu} {cells}} \\{{incubated}\mspace{14mu} {at}\mspace{14mu} 4{^\circ}\mspace{14mu} {C.\mspace{14mu} {for}}\mspace{14mu} 60\mspace{14mu} {minutes}}\end{matrix}} \times 100}$

The results are shown in Table 2, Table 3, and FIG. 13.

TABLE 2 FITC APC Mean Mean fluorescence fluorescence intensity intensityTemperature of Fluorescence Temperature of Fluorescence incubationintensity incubation intensity (° C.) ratio (° C.) ratio 4 37 (%) 4 37(%) #17 35.7 15.9 44.5 1384 1320 95.4 #26 29.8 16.5 55.4 844 816 96.7#37 51.0 28.5 55.9 2194 2155 98.2 #28 40.6 19.3 47.5 1746 1709 97.9 #3347.7 22.6 47.4 1882 1845 98.0 IgG2a 3.7 4.2 116.2 3 3.64 121.3

TABLE 3 Fluorescence intensity Species of primary ratio (%) antibodiesAPC FITC Example 5 Anti-ILT7 antibody #17 95.4 44.5 Anti-ILT7 antibody#26 96.7 55.4 Anti-ILT7 antibody #37 98.2 55.9 Anti-ILT7 antibody #2897.9 47.5 Anti-ILT7 antibody #33 98.0 47.4 Comparative Mouse IgG2a 121.3116.2 example 2

The fluorescence intensity of FITC is an indicator of the amount ofILT7-anti-ILT7 antibody immune complex remained on the cell surfaceafter incubation. The mean fluorescence intensity of FITC as to thecells incubated at 37° C. for 60 minutes fell to about 50% as comparedwith the cells incubated at 4° C.

On the other hand, APC fluorescence intensity is an indicator of theamount of ILT7-anti-ILT7 antibody immune complex presented on the cellsurface before incubation. ILT7-anti-ILT7 antibody immune complex isdetected regardless of whether it is present on the cell surface orincorporated into cells after incubation. In Example 5, APC fluorescenceintensity after the incubation in the case of incubation at 37° C. wasequivalent to that in the case of incubation at 4° C. It shows thatILT7-anti-ILT7 antibody immune complex may be present in any site oftarget cells even when the incubation is performed at eithertemperature. As mentioned above, it was found that the anti-ILT7monoclonal antibody evoked the internalization of ILT7 by the incubationat 37° C.

Comparative Example 2

Exactly the same operation was performed in the same manner as describedin Example 5 except that mouse IgG2a was used in place of anti-ILT7antibody. The results are shown in Example 5 as well as Table 2, Table3, and FIG. 13. In the case where the mouse IgG2a was used, any changesin the fluorescence intensity of FITC and APC were not observed, thus itwas found that the mouse IgG2a did not evoke the internalization ofILT7.

Example 6 Concerning the Structure of Mouse Anti-Human ILT7 MonoclonalAntibody [Sequences of Variable Regions]

A. Cloning of cDNA Encoding Variable Region of Mouse Anti-ILT7 AntibodyA-1) Concerning Hybridomas which Produce Mouse Anti-ILT7 Antibodies

The following hybridomas were used as hybridomas which produce mouseanti-ILT7 antibodies.

Hybridoma #11 (Accession number: FERM BP-10704)

Hybridoma #17 (Accession number: FERM BP-10705)

A-2) Isolation of the Total RNAs

The total RNAs were isolated from hybridomas described in A-1) using acommercially available kit “RNeasy Mini Kit” (Catalog number: 74106,manufactured by Qiagen) in accordance with the instruction attached tothe kit. In both cases, about 200 μg of the total RNAs was obtained from1×10⁷ hybridomas.

A-3) Amplification and Fragmentation of cDNA Encoding a Mouse HeavyChain Variable Region

cDNA encoding a mouse heavy chain variable region was amplified by5′RACE method using 5 μg of the total RNAs isolated as described in A-2.As for amplification, commercially available kit “5′RACE System forRapid Amplification of cDNA ENDs, Version 2.0 Kit” (Catalog number:18374-058, manufactured by Invitrogen) was used. It will be specificallydescribed as follows. First, a first strand cDNA was synthesized fromthe total RNAs obtained as described in A-2) by reverse transcriptase.The base sequences of antisense primers (GSP1) used at the time areshown in Table 4.

TABLE 4  Primers used for amplification of a gene encoding a mouse heavy chain variable region Used Names of SEQ hybridomas primers ID No.Sequence #11 Mu IgG3VH5RACE-GSP1 30 5′CCA TAG TTC CAT TTT ACA GTT ACC 3′ (24-mer) Mu IgG3VH5RACE-GSP2 31 5′GGG ACC AAG GGA TAG ACA GA 3′ (20-mer) Mu IgG2aVH5RACE-GSP1 32 5′TCC AGA GTT CCA GGT CAA GGT CAC 3′ #17 (24-mer) Mu IgG2aVH5RACE-GSP2 335′ GCC AGT GGA TAG ACC GAT GG 3′ (20-mer)

Subsequently, the total RNAs were degraded by RNaseH and the firststrand cDNA remained as a single strand was purified by low-meltingpoint agarose method (1.5%). Further, dC (i.e. nucleotide homopolymer)was attached to the 3′-terminus of the first chain cDNA using terminaldeoxynucleotidyl transferase (TdT). cDNA was amplified by PCR methodusing an anchor primers (SEQ ID NO: 34) having a nucleotide polymercomplementary to dC (anchor sequence) at 3′-terminus and antisenseprimers (GSP2) shown in Table 4. Further, the obtained PCR products wereused as templates. cDNA was amplified by Nested PCR method using AUAPprimer (SEQ ID NO: 35) and antisense primers (GSP2) shown in Table 4.Further, the PCR products were purified by low-melting point agarosemethod (1.5%).

Anchor primer for 5′RACE (SEQ ID NO: 34)5′-GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3′ (36-mer)AUAP primer gor 5′RACE (SEQ ID NO: 35)5′-GGC CAC GCG TCG ACT AGT AC-3′ (20-mer)A-4) Amplification and Fragmentation of cDNA Encoding Mouse Light ChainVariable Region

cDNA encoding a mouse light chain variable region was amplified from thetotal RNAs isolated as described in A-2) in the same manner as describedin A-3). The base sequences of primers used at the time is shown inTable 5. The obtained PCR products were purified by low-melting pointagarose method (1.5%).

TABLE 5 Primers used for amplification of a gene encoding amouse light chain variable region Used Names of SEQ hybridomas primersID No. Sequence #11, #17 Mu IgVL5RACE-GSP1 36 5′TTC ACT GCC ATC AAT CTT CCA CTT 3′ (24-mer)  Mu IgVL5RACE-GSP2 37 5′GAT GGA TAC AGT TGG TGC AGC 3′ (21-mer)A-5) Confirmation of Base Sequence of cDNA and Determination of CDRRegion

A heavy chain variable region obtained as described in A-3) and cDNAfragment of light chain variable region obtained as described in A-4)were cloned to pCR4 Blunt-TOPO vector using a commercially available kit“Zero Blunt TOPO PCR Cloning Kit” (Catalog number: 1325137, manufacturedby Invitrogen) in accordance with the instruction attached to the kit,which was then introduced into Escherichia coli competent cells to giveEscherichia coli transformant. The above-mentioned plasmid was obtainedfrom the transformant, then cDNA base sequence in the plasmid wasconfirmed using an automatic DNA sequencer “PCR-based ABI PRISM 3100Genetic Analyzer” (manufactured by Applied Biosystems). Correctsequences were extracted by excluding transcripts obtained from aninactive RNA due to frame shift and nonsense mutations around thecomplementarity-determining region (hereinafter referred to as “CDRregion”). Further, the homology of cDNA base sequence comprised in theplasmid was compared with Kabat database and sequences of the CDR regionand the variable region in respective variable regions were determined.Also, as for the hybridoma #37 produced in Example 4, sequences of theCDR region and the variable region in variable regions were determinedin the same procedure as described in A-1) to A-5) of Example 6 usinghybridoma #17. cDNA base sequences of the heavy chain variable regionsand light chain variable regions of the anti-ILT7 monoclonal antibodiesproduced by each hybridoma and amino acid sequences encoded by thesequences are shown in the following SEQ ID NOs.

Heavy chain variable region Light chain variable region #11 SEQ ID NO:38 SEQ ID NO: 40 (base sequence) (base sequence) SEQ ID NO: 39 SEQ IDNO: 41 (amino acid sequence) (amino acid sequence) #17 SEQ ID NO: 42 SEQID NO: 44 (base sequence) (base sequence) SEQ ID NO: 43 SEQ ID NO: 45(amino acid sequence) (amino acid sequence) #37 SEQ ID NO: 46 SEQ ID NO:48 (base sequence) (base sequence) SEQ ID NO: 47 SEQ ID NO: 49 (aminoacid sequence) (amino acid sequence)

[Confirmation of Isotype of Constant Region]

As for the hybridoma culture supernatant, the isotype of the constantregion of the produced monoclonal antibody was confirmed using acommercially available mouse monoclonal antibody isotyping kit (Catalognumber: MMT1, manufactured by Serotec Product). The heavy chain constantregion of mouse anti-human ILT7 antibody #11 was Igγ3 and the lightchain constant region was Igκ. Further, each of the heavy chain constantregions of mouse anti-human ILT7 antibody #17 and mouse anti-human ILT7antibody #37 was Igγ2a and each of the light chain constant region wasIgκ.

Example 7 Production of Chimeric Antibodies

A. Cloning of cDNA Encoding Human IgG Constant Region

Human IgG1 heavy chain constant region and human Ig kappa light chainconstant region were selected from cDNA library of human IPCs. Then, theselected regions were cloned to pCR4 Blunt-TOPO vector using acommercially available kit “Zero Blunt TOPO PCR Cloning Kit” (Catalognumber: 1325137, manufactured by Invitrogen) in accordance with theinstruction attached to the kit, which was then introduced intoEscherichia coli competent cells to give Escherichia coli transformant.The above-mentioned plasmid was obtained from the transformant, thencDNA base sequence in the plasmid was confirmed using an automatic DNAsequencer “PCR-based ABI PRISM 3100 Genetic Analyzer” (manufactured byApplied Biosystems).

B. Ligation of Variable Region with Constant Region and Cloning

The cDNA encoding the heavy chain constant region obtained as describedin A and the cDNA encoding the heavy chain variable region obtained asdescribed in A-5 of Example 6 was used, respectively. Both DNAs have aregion in which a base sequence of DNA is overlapped. Then,double-stranded DNA was obtained by the overlap extension method usingthe region. Specific process is as follows.

C-1) Preparation of cDNA Encoding Heavy Chain of Chimeric ILT7 Antibody

The “plasmid with cDNA encoding heavy chain variable regions of #11 and#17” which was obtained as described in A-5) was digested withrestriction enzymes NotI and XbaI, which was purified by the agarose gelmethod (1.5%). The resulting products were dissolved in each TE bufferwith the following composition so as to be 100 pmol/μL to prepare asolution of the cDNA fragment encoding the heavy chain variable region.

TE Buffer: 10 mM Tris-HCl 1 mM EDTA

pH 7.5 to 8.0

Further, the “plasmid with cDNA encoding the heavy chain constantregion” obtained as described in B was treated in the same manner asdescribed above to prepare 100 pmol/μL of solution. Subsequently, bothsolutions were mixed, and then both of the overlap regions werehybridized by first keeping them at 70° C. for 10 minutes and nextkeeping them at 37° C. for 5 minutes. Thereafter, cDNA was amplified byPCR method and the obtained cDNA was digested with restriction enzymesNotI and XbaI, which was purified by the low-melting point agarose gelmethod (1.5%).

C-2) Preparation of cDNA Encoding Light Chain of Chimeric ILT7 Antibody

The cDNA encoding the light chain constant region obtained as describedin A and the cDNA encoding the light chain variable region obtained asdescribed in A-5 of Example 6 was used, respectively. cDNA encoding thelight chain of chimeric ILT7 antibody was obtained in the same manner asdescribed in C-1) using these cDNAs.

C-3) Cloning

cDNA obtained as described in C-1) was cloned to plasmid vectorpcDNA3.1-Zeocin (manufactured by Invitrogen) using NotI and XbaI ascloning sites to produce a chimeric ILT7 antibody heavy chain expressionvector. Further, cDNA obtained as described in C-2) was cloned toplasmid vector pcDNA3.1-hygromycin (manufactured by Invitrogen) usingNotI and XbaI as cloning sites to produce a chimeric ILT7 antibody lightchain expression vector. Names of each vector are shown in Table 6.

TABLE 6 Names of plasmid vectors Chimeric ILT7 antibody heavy ChimericILT7 antibody light chain for expression chain for expression #11pcDNA-#11VH pcDNA-#11VL #17 pcDNA-#17VH pcDNA-#17VL

D. Expression of Chimeric ILT7 Antibody D-1) Transient Transformation

1 μg of chimeric ILT7 antibody heavy chain expression vector and 1 μg ofchimeric ILT7 antibody light chain expression vector, which wereobtained as described in C-3), were co-transfected to 293T cells usingeffectine transfection kit (Catalog number: 301427, manufactured byQiagen). Thereafter, the resulting products were cultured at 37° C.using 2% Low IgG FBS-added DMEM culture medium with the followingcomposition.

2% Low IgG FBS-added DMEM culture medium:DMEM culture medium (Catalog number: D5796, manufactured by Sigma)2% Low IgG FBS (Catalog number: SH30151.03, manufactured by HyClone)

2 mM L-Glutamin 100 U/ml Penicillin

100 μg/ml StreptomycinpH 7.2 to pH 7.4

After introduction of vectors, the resulting medium was cultured for 96hours and the culture supernatant was collected. Then, the cellfragments were removed by centrifugation to give a crude antibodysolution.

D-2) Homeostasis Transformation

1 μg of chimeric ILT7 antibody heavy chain expression vector and 1 μg ofchimeric ILT7 antibody light chain expression vector, which wereobtained as described in C-3), were co-transfected to YB 2/0 cells(cells derived from rat myeloma, ATCC#CRL-1622) using effectinetransfection kit (Catalog number: 301427, manufactured by Qiagen). Amongthe used plasmid vectors, the vector for the heavy chain expression is amarker for Zeocin resistance and the vector for the light chainexpression is a marker for hygromycin resistance. Therefore, cells intowhich both vectors were introduced can be grown in a culture medium towhich Zeocin and hygromycin are added at the same time. Then, the cellswere cultured in RPMI culture medium to which Zeocin and hygromycin wereadded and a resistant strain was selected.

Zeocin-Hygromycin-Added RPMI Culture Medium:

RPMI1640 culture medium (Catalog number: R8758, manufactured by Sigma)

10% FBS

0.01 M HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid)1 mM Sodium pyruvate

2 mM L-Glutamine 100 U/ml Penicillin

100 μg/ml Streptomycin55 μM 2-mercaptoethanol0.5 mg/ml Zeocin0.5 mg/ml HygromycinpH 7.2 to pH 7.4

Three days after that, the amount of antibody production in the culturesupernatant was determined by the ELISA method. ILT7 chimeric-antibodyproducing cell line with a high expression level and cells sufficientlyincreased was selected. Furthermore, a single cloning of the selectedcell lines was performed by the cell sorter method to obtain thefollowing cell lines.

#11 ILT7 chimeric-antibody-producing cell line: #11-5 cell line and#11-16 cell line#17 ILT7 chimeric-antibody-producing cell line: #17-24 cell line

The above-mentioned cell lines (#11-5 cell line, #11-16 cell line, and#17-24 cell line) were respectively cultured in 5% FBS-added RPMIculture medium with the following composition. The incubationtemperature and incubation time were set to 37° C. and 96 hours,respectively.

5% FBS-Added RPMI Culture Medium:

RPMI1640 culture medium (Catalog number: R8758S, manufactured by Sigma)

5% FBS 0.01 M HEPES

1 mM Sodium pyruvate

2 mM L-Glutamin 100 U/ml Penicillin

100 μg/ml Streptomycin55 μM 2-mercaptoethanolpH 7.2 to pH 7.4

The culture supernatant was collected and then the cell fragments wereremoved by centrifugation to give a crude antibody solution.

E. Purification of Antibodies

Each of the crude antibody solutions obtained as described in D-1 andD-2 was purified by protein A affinity column (rProtein A Sepharose FF,Catalog number: 17-1279-01, manufactured by Amershram Pharmacia).Purification conditions are as follows. Affinity purification wascarried out using PBS (−) buffer with the following composition as anadsorption buffer and 0.1M sodium citrate buffer (pH 3) as an elutionbuffer in accordance with the attached instruction manual. 1 M Tris-HCl(pH 8.0) was added to the eluted fractions to adjust the pH to around7.2. The ODs at 450 to 620 nm were measured and then wells showingpositive reaction were selected. With reference to the concentration ofpurified antibodies, the absorbance at 280 nm was determined andcalculated based on 1.38 OD/mg/ml. Relationships among chimeric ILT7antibodies obtained, hybridomas from which the variable region gene wasderived, and host cells were summarized in Table 7.

PBS (−) buffer:0.2 g/L Monopotassium dihydrogen phosphate0.2 g/L Potassium chloride8 g/L Sodium chloride1.15 g/L Disodium monohydrogen phosphate anhydrous

TABLE 7 Produced chimeric antibodies Names of produced chimeric UsedForm of Introduced antibodies hybridomas transformation cells #11 ILT7chimeric antibody #11 Transient 293T #17 ILT7 chimeric antibody #17manner #11-5 ILT7 chimeric antibody #11 Homeostasis YB 2/0 #11-16 ILT7chimeric #11 antibody #17-24 ILT7 chimeric #17 antibody

cDNA base sequences and amino acid sequences of the heavy and lightchains of the produced chimeric antibodies are shown below,respectively. In each amino acid sequence, the amino acid sequence fromN-terminus to −1 is a signal sequence and the amino acid sequence from 1to C terminus is an amino acid sequence of a mature protein. That is,heavy and light chains, which constitute these chimeric antibodies,consist of the amino acid sequence from 1 to C terminus of each of thefollowing amino acid sequences.

Heavy chain Light chain #11 SEQ ID NO: 50 SEQ ID NO: 52 (base sequence)(base sequence) SEQ ID NO: 51 SEQ ID NO: 53 (amino acid sequence) (aminoacid sequence) #17 SEQ ID NO: 54 SEQ ID NO: 56 (base sequence) (basesequence) SEQ ID NO: 55 SEQ ID NO: 57 (amino acid sequence) (amino acidsequence)

INDUSTRIAL APPLICABILITY

The present invention provided the immunogen useful in producing theantibody specifically recognizing human ILT7 and the method forproducing anti-ILT7 antibody using the immunogen. The antibodyspecifically recognizing human ILT7 of the present inventionspecifically recognizes ILT7 under the presence of ILT family.Therefore, the antibody of the present invention can be used for thedetection and isolation of human ILT7. For example, the localization ofILT7 can also be analyzed using the antibody of the present invention.It is considered that ILT7 is a molecule closely related to thedifferentiation and function of IPCs or dendritic cells. Therefore, theantibody, which recognizes ILT7 with high specificity, is useful for theanalysis of function of IPCs or dendritic cells. IPC-like (having thecharacteristic in which BDCA-2 is expressed) cancer cells are known(Chaper of L et al. Eur. J. Immunol. 34; 418-426, 2004, Maeda T et al.,Int. J. Hematol. 81; 148-154, 2005). Confirmation of the expression ofILT7 in these cells may allow for the diagnosis of cancer and atherapeutic agent.

In the case of autoimmune diseases, for example, the deep relationshipbetween IFNα produced by IPCs and the development of psoriasis, which isa skin disease, is pointed out (Nestle F O et al., J. Exp. Med. 202,135-143, 2005). Therefore, the severity of psoriasis can be examined byidentifying IPCs in the skin tissue of psoriasis patients, i.e. inbiopsy specimens using the anti-ILT7 antibody.

It is known that the development of AIDS in HIV-infected patients iscorrelated with the number of IPCs. Namely, lots of IPCs have beenobserved in patients who do not show symptoms and the reduction in IPCshas been observed in the onset (Soumells V. et al., Blood 98; 906-912,2001). Therefore, it is effective in predicting the prognosis of virusinfection, such as HIV.

For example, ILT7 is a molecule which is expressed specifically in humanIPCs. Therefore, the anti-ILT7 antibody of the present invention can beused to detect, identify, or isolate IPCs. IPCs are cells which producemost of the type 1 interferon. Therefore, the detection, identification,or isolation is an important objective in diagnosis and study ofdiseases that involve type 1 interferon. As such diseases, variousautoimmune diseases and infections that interferon is involved in theformation of the pathological condition may be illustrated.

Additionally, the anti-ILT7 antibody of the present invention has theinhibitory effect on the activity of IPCs. Therefore, the activity ofIPCs can be inhibited by using the anti-ILT7 antibody of the presentinvention. Furthermore, the diseases that involve type 1 interferon canbe treated by inhibiting the activity of IPCs. Specifically, theanti-ILT7 antibody of the present invention is useful for variousautoimmune diseases and infections that interferon is involved in theformation of the pathological condition. Particularly, since theanti-ILT7 antibody has a high specificity, it can remove IPCsefficiently.

1-31. (canceled)
 32. A monoclonal antibody which binds to human ILT7,binds to none of human ILT1, human ILT2 and human ILT3, and suppressesinterferon-α (IFNα) production from ILT7-expressing cells, or a fragmentcomprising its antigen binding region.
 33. The monoclonal antibodyaccording to claim 1, wherein the ILT7 expressing cell is an interferonproducing cell (IPC).
 34. The monoclonal antibody according to claim 1or 2, wherein the cell is CD123 positive or BDCA-2 positive.
 35. Themonoclonal antibody according to any one of claims 1 to 3, wherein thecell is CD3 negative, CD14 negative, CD16 negative, CD19 negative, CD20negative, and CD56 negative.
 36. The monoclonal antibody according toany one of claims 1 to 4, wherein the monoclonal antibody is produced bya method comprising the following steps of: (1) administering to anon-human animal a cell retaining the following (a) and (b) in anexpressible manner: (a) an exogenous polynucleotide encoding an aminoacid sequence comprising an extracellular domain of human ILT7; and (b)an exogenous polynucleotide encoding Fc receptor γ-chain (FcRγ); (2)producing and selecting an antibody producing cell which produces anantibody which binds to human ILT7 from the antibody producing cell ofthe non-human animal; and (3) culturing the cell so selected andrecovering a monoclonal antibody from the culture.
 37. The monoclonalantibody according to claim 5, wherein the cell is an animal cell; andoptionally wherein the cell is a human-derived cell; and furtheroptionally wherein the human-derived cell is a 293T cell.
 38. Themonoclonal antibody according to any one of claims 1 to 4, wherein thefragment comprising its antigen binding region is Fab, F(ab′)2, singlechain Fv, or diabody.
 39. A method for detecting an interferon producingcell, comprising the steps of: contacting the monoclonal antibody of anyone of claims 1 to 7 with a test cell; and detecting the monoclonalantibody or the fragment comprising its antigen binding region which isbound to the cell.
 40. An interferon α (IFNα)-suppressing agentcomprising the monoclonal antibody according to any one of claims 1 to 7as an active ingredient.